Abstract:
A seed-specific expression vector and its construction methods and applications are disclosed. A fusion protein expression cassette consisting of  Arachis hypogaea  oleosin gene-apolipopoprotein A-I Milano  (A-IM) gene driven by  Brassica napus  oleosin gene promoter is inserted between the HindIII and SacI sites of a plant binary expression vector pBI121, obtaining the plant expression vector pBINOA of the invention. In addition, a method for producing apolipoprotein A-I Milano  is provided, in which the expression vector is used to transform oil sunflower which is used as a plant bioreactor. The method can not only improve the yield of apolipoprotein A-I Milano , but also greatly reduce production costs, and is suitable for industrial production.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention is directed to a seed-specific expression vector and its construction methods and applications, and in particular to a seed-specific expression vector and its construction methods and a method for producing apolipoprotein A-I- Milano  (AIM) in oil sunflower with this vector. 
       BACKGROUND OF THE INVENTION 
       [0002]    Cardiovascular disease (CVD) is the leading cause of human deaths worldwide. It is estimated that, by 2015, approximately 20 million people will die from cardiovascular disease (CVD). Numerous cardiovascular diseases (CVDs) (such as myocardial infarction and apoplexy) are the leading complications of atherosclerosis (AS). So far, the pathogenesis of atherosclerosis has not been fully understood. Abnormal lipid metabolism is one of the main risk factors that cause this disease. High-level of low density lipoprotein (LDL) and low-level of high density lipoproteins (HDL) are the two most important risk factors. Traditional strategies of treatment are to reduce the content of total cholesterol (TC) and low density lipoproteins cholesterol (LDL-C) in blood plasma. Statins are the preferred lipid-lowering drug at present. However, they cannot eliminate existing plaques deposited on artery wall, or fundamentally cure atherosclerosis (AS). More and more scientists in different countries have turn to the other risk factor, low-level of high density lipoprotein (HDL). Epidemiological studies indicate that, the level of high density lipoproteins (HDL) in blood plasma is in negative correlation to the incidence of coronary diseases. It is believed that high density lipoprotein contributes to the prevention of atherosclerosis. Treating atherosclerosis through improving the level of high density lipoprotein is a new approach for treating acute coronal atherosclerosis diseases that is emerging in pharmaceutical industry. It is called high density lipoprotein targeted therapy. Apolipoprotein A-I (apo A-I) is the main proteic component of high density lipoproteins. Apolipoprotein A-I is synthesized in liver and small intestine. The primary translation product is the preproprotein (preproapo A-I) containing 267 amino acid residues. Preproprotein is then processed into the proprotein (proapo A-I), through the cleavage of an octadeca peptide by signal peptidase. The proapo is secreted and processed into mature plasma apolipoprotein A-I through cleavage of a hexapeptide (Arg-His-Phe-Trp-Gln-Gln) by specific extracellular converting enzymes. The mainly mechanism of action of apolipoprotein A-I is to promote the cholesterol efflux, antioxidation, and to decrease platelet aggregation. 
         [0003]    Apolipoprotein (apolipoprotein A-IM, apoA-IM) is a natural mutant of apolipoprotein A-I (Arg173-Cys). Compared with apolipoprotein A-I, the loss of Arg173 leads to the reduction of content of α-helix and the enhancement of the capability to bind lipid. Apolipoprotein A-I- Milano  tends to form a dimer (A-IM/A-IM). This dimer stimulates the reverse transport of cholesterol, and thus the clearance of cholesterol, more efficiently than apolipoprotein A-I. Compared with apolipoprotein A-I, apolipoprotein A-I- Milano  more efficiently decrease the oxidation of low density lipoprotein. At present, apolipoprotein A-I- Milano  is the only pharmaceutical protein that is shown to remove the thrombus deposited on artery wall, with broad application prospect. 
         [0004]    It is reported in the Journal of the American Medical Association (JAMA) recently that, apolipoprotein A-I- Milano  effects changes of artery atherosclerosis lesion with unprecedentedly speed and amplitude and little side effect. With various application prospects, it has become the focus of pharmaceutical research and industrial competition worldwide. Pfizer, the largest pharmaceutical company in the world, estimates that any drug reversing artery plaque may be a billion dollar business. Therefore, the development of apolipoprotein A-I- Milano  will definitely bring about enormous economical and social benefits, as well as enhance the competitive strength in the field of drug development against cardiovascular diseases and atherosclerosis diseases. 
         [0005]    In addition, data obtained from small-scale clinical trials reveal that the clinical dosage of apolipoprotein A-I is 5-6 g per treatment course. The high therapeutic dosage of apolipoprotein A-I and the high prevalence of atherosclerosis suggest huge market demand and also an opportunity for the development of apolipoprotein A-I- Milano . At present, apolipoprotein A-I- Milano  is produced by Eperion, US by means of biosynthesis, which is of high cost and low yield and undesirable for large-scale production. The recombinant expression of the protein in bacterial system is generally attractive. However, the yield is low, and  Escherchia coli  endotoxin tends to form tight complex with apolipoprotein A-I- Milano . Besides, the protein purification method is expensive and poor in safety. Therefore, there is the need for a method of producing apolipoprotein A-I- Milano  with high yield and efficiency. 
         [0006]    Plant bioreactor, also called molecular medicine farming, refers to the large-scale production of heterologous proteins of importance and commercial value, especially medical proteins used for the treatment or diagnosis of diseases, by a plant biological system. Mammalian antibodies were successfully expressed in transgenic plants for the first time in 1989. Both the heavy and light chains were expressed and correctly assembled in transgenic tobacco, demonstrating for the first time the possibility to use plant as a bioreactor. Since then, researches directed to transgenic plants have been rising. Many other medical proteins have been expressed in different plants sooner or later, such as hirudin, interferon, human albumin, and functional antibodies. Plants already used in plant bioreactor research include tobacco,  Arabidopsis thaliana , soybean, wheat, rice, rape, potato and tomato, etc. 
         [0007]    SemBioSys Genetic, Inc, a Canadian biotechnology company developing protein drug combinations for metabolic and cardiovascular diseases, filed a patent application in China (CN1906296A) regarding the method for producing apolipoprotein A-I and apolipoprotein A-I- Milano  with transgenic  Carthamus tinctorius  and  Arabidopsis thaliana , in which a chimeric nucleic acid construct is introduced into  Arabidopsis thaliana  or  Carthamus tinctorius . Apolipoprotein A-I and apolipoprotein A-I- Milano  is expressed in seeds upon seed setting.  Arabidopsis thaliana  is an annual or biennial herb. It has the smallest genome among all plants. Due to its high generic homozygosity, high mutation rates may be achieved upon physical or chemical treatments, providing various metabolic deficiency phenotypes. Thus  Arabidopsis thaliana  represents a good material for genetics research, and is called “the fruit fly of the plant world”. Though  Arabidopsis thaliana  is widely used in experimental contexts, it is not utilized in large-scale production.  Carthamus tinctorius  is an annual herb. Its seed can be used for oil extraction, and thus it is an important oil crop. It is distributed in the temperate zone. In China, it is mainly distributed in the Northwest (in particular Xinjiang and Tibet), and then North China and Northeast regions.  Carthamus tinctorius  suffers from the disadvantage of relatively low yield per mu (120-150 kg) and suboptimal oil content of the achene (34˜55%), resulting in low productivity of the end product protein and a high cost. 
         [0008]    Therefore, there is still the need for a method for producing apolipoprotein A-I and apolipoprotein A-I- Milano  with stable and high yield, low cost, and simple procedures. 
       BRIEF DESCRIPTION OF THE INVENTION 
       [0009]    The inventor, upon extensive investigation and creative work, accomplished the invention by stably and high-efficiently producing apolipoprotein A-I and apolipoprotein A-I- Milano  through the construction of a specific expression vector and utilizing oil sunflower as bioreactor. 
         [0010]    The present invention is directed to a method for producing apolipoprotein A-I or apolipoprotein A-I- Milano  by recombinant DNA technology, in particular a method for producing apolipoprotein A-I or apolipoprotein A-I- Milano  using oil sunflower as the host. Specifically, the invention involves the expression of the gene of a fusion protein consisting of  Arachis hypogaea  oleosin and apolipoprotein A-I or apolipoprotein A-I- Milano  in oil sunflower oil body, thereby producing the important drugs apolipoprotein A-I and apolipoprotein A-I- Milano  preferably apolipoprotein A-I- Milano , used for treating atherosclerosis and the related cardiovascular diseases. 
         [0011]    In one aspect, the invention provides a seed-specific expression vector comprising apolipoprotein A-I- Milano  gene fused with  Arachis hypogaea  oleosin gene or apolipoprotein A-I gene fused with  Arachis hypogaea  oleosin gene, preferably apolipoprotein A-I- Milano  gene fused with  Arachis hypogaea  oleosin gene, in which the promoter of the said vector is the  Brassica napus  oleosin gene promoter. The above vector is used for producing apolipoprotein A-I or apolipoprotein A-I- Milano , preferably apolipoprotein A-I- Milano  in oil sunflower. 
         [0012]    In another aspect, the invention provides a method for the construction the above high-efficient seed-specific expression vector, including the following steps: 
         [0013]    1) Isolating and cloning of  Brassica napus  oleosin gene promoter and  Arachis hypogaea  oleosin gene; 
         [0014]    2) Designing and synthesizing an apolipoprotein A-I- Milano  gene or apolipoprotein A-I gene according to the codon preference of the plant; 
         [0015]    3) Constructing a plant expression vector in which the fusion of  Arachis hypogaea  oleosin gene with apolipoprotein A-I- Milano  or apolipoprotein A-I gene is driven by  Brassica napus  oleosin gene promoter. 
         [0016]    The details of the steps are explained as follows: 
         [0017]    1) Isolating and cloning of  Brassica napus  oleosin gene promoter and  Arachis hypogaea  oleosin gene: The 20 kD oleosin gene promoter is amplified by PCR from  Brassica napus  genome DNA, and cloned into pUC19 (purchased from MBI), obtaining a recombinant plasmid pUCN. The  Arachis hypogaea  oleosin gene lacking the stop codon is amplified by PCR using  Arachis hypogaea  genome DNA as template. The specific rape variety may be one that is published or used in the art, such as Qingyou 14, Hufeng 101, cold-resistance king of high oil, Early Oil 100-Day, Qingyou 2, etc., preferably Qingyou 14. The  Brassica napus  oleosin gene promoter may be cloned between the appropriate sites of pUC19, and preferably between the HindIII and BamHI sites of pUC19. The  Arachis hypogaea  variety may be one that is already published or used in the art, such as Jihua 4, Jiyou 7, Baisha, Luhua 11, Haihua, Fenghua 1, etc., preferably jihua 4. 
         [0018]    2) Designing and synthesizing an apolipoprotein A-I- Milano  gene or apolipoprotein A-I gene according to the codon preference of the plant: This is to optimize apolipoprotein A-I- Milano  or apolipoprotein A-I gene (preferably the former) according to the codon usage of  Helianthus  animus. All codons with the usage frequency of less than 10% shall be regarded as rare codons and thus abolished, while the remaining codons shall be optimized according to the frequency of  Helianthus annuus  codon usage. The molecular weight of gene before optimization is 451.4. The sequence identity of the sequences before and after optimization is higher than 60%, preferably higher than 65%, even preferably higher than 72%. The preferable molecular weight of gene after optimization is 451.3. For the usage frequency of  Helianthus annuus  codons, reference may be made to http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=4232. 
         [0019]    3) Constructing a plant expression vector in which the fusion of  Arachis hypogaea  oleosin gene with apolipoprotein A-I- Milano  or apolipoprotein A-I gene is driven by  Brassica napus  oleosin gene promoter: The fusion gene of  Arachis hypogaea  oleosin gene with apolipoprotein A-I- Milano  gene or apolipoprotein A-I gene is constructed by overlapping PCR. Preferably,  Arachis hypogaea  oleosin gene is fused with apolipoprotein A-I- Milano  gene, obtaining the fusion gene Ole/apoA-I M . The fusion gene is linked into pUCN, preferable between the BamHI and SacI sites, obtaining recombinant plasmid pUCNOA. The recombined plasmid pUCNOA is subjected to double digestion, preferably with HindIII and SacI, to recover the exogenous fragment of 2202 bp. The exogenous fragment is subsequently inserted between the HindIII and SacI of pBI121, a binary plant expression vector commonly used in plant transgenic engineering, obtaining pBINOA, the plant expression vector in which the fusion gene of  Arachis hypogaea  oleosin with apolipoprotein A-I- Milano  is driven by  Brassica napus  oleosin gene promoter, or in which the fusion gene of  Arachis hypogaea  oleosin gene with apolipoprotein A-I is driven by  Brassica napus  oleosin gene promoter. 
         [0020]    In another aspect, the invention provides a method for producing apolipoprotein A-I- Milano  or apolipoprotein A-I with the above seed-specific plant expression vectors, including the following steps:
       1) introducing the above construction expression vectors into an explant of a receptor plant;   2) cultivating the above receptor plant materials into a complete plant and obtain seeds thereof;   3) isolating apolipoprotein A-I- Milano  or apolipoprotein A-I from the seeds.       
 
         [0024]    Preferably, the receptor plant is oil sunflower. Preferably, apolipoprotein A-I- Milano  is produced. 
         [0025]    The specific procedures include the following details. 
         [0026]    1) A seed-specific plant expression vector carrying apolipoprotein A-I- Milano  gene or apolipoprotein A-I gene is introduced into the explant of a oil sunflower restoring line. The method for introducing the seed-specific plant expression vector into the restoring line of oil sunflower may be a conventional introduction method in the art, including but not limited to gene gun bombardment, pollen-tube pathway, ovary injection, and  Agrobacterium -mediated transformation, preferably  Agrobacterium -mediated transformation. In  Agrobacterium -mediated transformation, the seed-specific plant expression vector carrying apolipoprotein A-I- Milano  gene or apolipoprotein A-I gene is introduced into  Agrobacterium , which mediates the transformation of the explants of the restoring line of oil sunflower. The explants include shoot apexes excised from sterile seedling, cotyledon, cotyledon node, and seedlings with one cotyledon detached. Preference is made to seeding plant stripped of one cotyledon. 
         [0027]    2) Resistance seedling is obtained through resistance selection of the regenerated plants obtained after transgenesis, and is transplanted into greenhouse after rootage for cultivation until maturity to harvest seeds. The resistance seedling is transplanted into greenhouse after taking root for vermiculite and nutritional soil mixture cultivation. PCR test and southern blotting test shall be conducted during the Seedling Stage. Western blotting test shall be conducted after harvest against the fusion protein of oleosin and apolipoprotein A-I- Milano ; 
         [0028]    3) The seed containing apolipoprotein A-I- Milano  or apolipoprotein A-I is ground in buffer solution. The oil body is separated from other components of the seed by centrifugation and washed. Apolipoprotein A-I- Milano  or apolipoprotein A-I is released from the oil body surface through digestion, purified by HPLC, and subjected to identification. 
         [0029]    In the vector and method of the invention,  Brassica  lupus oleosin promoter is used. Experimental research indicates that this promoter can greatly improve the expression efficiency of apolipoprotein A-I- Milano  gene. Preferably, Kozak consensus sequence may be positioned near the initiator codon of oleosin gene, further improving the expression efficiency. 
         [0030]    In the vector and method of the invention, the apolipoprotein A-I- Milano  or apolipoprotein A-I is expressed as fusion protein with oleosin. The protein of interest is specifically expressed in transgenic plants in the oil body as fusion with oleosin. Taking advantage of the hydrophobic/lipophilic characteristic of the oil body, the seeds of the transgenic plant is subjected to grind, extraction, centrifugation, and recovery of the upper oil phase, thereby separating the fusion protein from other components in the cell. More than 90% of seed proteins can be removed. Preferably, a thrombin recognition site is positioned between oleosin and apolipoprotein A-I- Milano  or apolipoprotein A-I for releasing apolipoprotein A-I- Milano  from oil body, thereby simplifying the purification process of the expression product and improving the purification efficiency. The preferred oleosin is  Arachis hypogaea  oleosin. The fusion expression of  Arachis hypogaea  oleosin and apolipoprotein A-I- Milano  or apolipoprotein A-I is optimal in terms of quality and quantity. 
         [0031]    In the vector and method of this invention, in order to improve the expression efficiency of apolipoprotein A-I- Milano  gene or apolipoprotein A-I gene, the apolipoprotein A-I- Milano  gene or apolipoprotein A-I gene is optimized according to apolipoprotein A-I- Milano  or apolipoprotein A-I gene sequence, the preference of codon usage of  Helianthus annuus  and GC content, and is fully synthetic. 
         [0032]    In the methods of apolipoprotein A-I- Milano  or apolipoprotein A-I production disclosed by this invention, the preferable plant bioreactor is oil sunflower. As an important oil crop in China, oil sunflower has a long planting history and irreplaceable advantages relative to other crops. With high yield and as a drought tolerance crop, oil sunflower can be planted in severe environment such as alkali soils, arid areas, and even in deserts. It is therefore suitable for large-scale planting. The planting of oil sunflower does not conflict with alimentary crops, and is beneficial in terms of improving the utilization of mountain ridges and dry and unfruitful area, alleviating the insufficiency of cultivated land. Therefore, it is particularly beneficial in China to use oil sunflower as bioreactor for the large-scale production of apolipoprotein A-I- Milano . A most significant advantage is the greatly improved production efficiency and productivity achieved by oil sunflower as bioreactor, compared with prior art methods using  Carthamus tinctorius  as the bioreactor for the production of apolipoprotein A-I- Milano  or apolipoprotein A-I. 
         [0033]    The following advantages are achieved by the method of producing apolipoprotein A-I- Milano  of the invention. 
         [0034]    1. The heterologous protein expressed in plant is similar to the protein expressed in mammals and can be correctly fold. This is of particular importance for the production of medical proteins with in vivo activity. 
         [0035]    2. The apolipoprotein A-I- Milano  produced in plant bioreactor is safer, because it avoids the contamination of  E. coli  endoxin or pathogens. 
         [0036]    3. The oil body expression system of transgenic plant used for expressing apolipoprotein A-I- Milano  greatly simplifies the purification process, reduces cost, and facilitates the industrialization, compared with  Arabidopsis thaliana  and  Carthamus tinctorius  systems already adopted by SemBioSys Genetics. 
         [0037]    4. The seed-specific plant expression vector and preparation method introduced by this invention can greatly improve the expression quantity of apolipoprotein A-I- Milano  or apolipoprotein A-I, which can reach 1.5% of the total protein content of seed. 
         [0038]    5.  Agrobacterium -mediated transformation is used, which not only reduce cost and improves transformation efficiency, but also improves the genetic stability of the transgenic plant. 
         [0039]    This invention utilizes transgenic technology to develop a high expression efficiency plant bioreactor. The resultant product, apolipoprotein A-I- Milano  or apolipoprotein A-I, is an potent drug for the treatment of cardiovascular diseases and atherosclerosis diseases. 
       DEFINITIONS 
       [0040]    Unless specially defined otherwise, all terms referred in this invention shall have the common meanings in the field, wherein the meaning of abbreviations are provided as follows: 
         [0041]    LDL: Low density lipoprotein (LDL) 
         [0042]    HDL: High density lipoproteins (HDL) 
         [0043]    TC: Total cholesterol (TC) in blood plasma 
         [0044]    LDL-C: Low density lipoproteins cholesterol (LDL-C) 
         [0045]    apoA-I: Apolipoprotein A-I 
         [0046]    apoA-IM: Apolipoprotein A-I- Milano  (AIM) 
         [0047]    A-IM/A-IM: apolipoprotein A-I- Milano  dimer 
         [0048]    pUC19: a common  E. coli  cloning vector, obtained from MBI 
         [0049]    pUC121: a common plant expression vector in plant transgenic engineering 
         [0050]    pUCN: pUC19 vector carrying  Brassica napus  oleosin promoter (NOP) inserted between the HindIII and BamHI sites 
         [0051]    Ole/apoA-IM: Fusion gene of  Arachis hypogaea  oleosin with apolipoprotein A-I- Milano    
         [0052]    pUCNOA: pUC19 vector carrying the fusion gene of  Brassica napus  oleosin gene promoter (NOP),  Arachis hypogaea  oleosin gene and apolipoprotein A-I- Milano , inserted between the HindIII and SacI sites 
         [0053]    pBINOA: pBI121 vector carrying the fusion gene of  Brassica napus  oleosin promoter (NOP),  Arachis hypogaea  oleosin gene and apolipoprotein A-I- Milano , inserted between the HindIII and SacI sites. 
     
    
     
       DESCRIPTION OF FIGURES 
         [0054]      FIG. 1  Schematic Drawing of the Seed-specific plant expression vector pBINOA; 
           [0055]      FIG. 2  Schematic Drawing of the Construction Process of Seed-specific plant expression vector pBINOA; 
           [0056]      FIG. 3  pUCNvector Restriction Enzyme Digestion Identification and PCR Detection; 
           [0057]      FIG. 4  Construction of Ole/apoA-IM Fusion gene; 
           [0058]      FIG. 5  pUCNOAvector Restriction Enzyme Digestion Identification; 
           [0059]      FIG. 6  Restriction Enzyme Digestion Identification of Seed-specific Plant Expression Vector pBINOA; 
           [0060]      FIG. 7  PCR Detection of npt II Gene in Transgenic Oil Sunflower; 
           [0061]      FIG. 8  PCR Detection of apolipoprotein A-I- Milano  Gene in Transgenic Oil Sunflower; 
           [0062]      FIG. 9  PCR-Southern Blotting Results of Transgenic Oil Sunflower; 
           [0063]      FIG. 10  Western Detection of the oleosin-Apolipoprotein A-I- Milano  Fusion Protein in Transgenic Oil Sunflower Kernel-Oil Body; 
           [0064]    
         FIG. 11 
       
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0065]    The following embodiments are provided for further description of this invention, and are not construed as limiting to the scope of the invention. Given the present disclosure, alterations may be made to this invention without departing from the spirit of this invention. All these alterations are within the scope of the present invention. 
         [0066]    Unless otherwise specified, the methods referred to in the following embodiments are practiced according to general practice in this field. 
       Example 1 
     Seed-Specific Plant Expression Vector 
       [0067]      Brassica napus  oleosin gene promoter (NOP) was amplified by PCR, inserted into pUC19 between the HindIII and BamHI sites, obtaining pUCN. Apolipoprotein gene was designed according to apolipoprotein A-I- Milano  (AIM) gene sequence and the codon usage of  Helianthus annuus , synthetically produced, and inserted at the 3′ end of the  Arachis hypogaea  oleosin gene (Ole), obtaining the fusion gene of  Arachis hypogaea  oleosin and apolipoprotein A-I- Milano . Thrombin cleavage site was added between the  Arachis  hypogaea oleosin gene and the apolipoprotein A-I- Milano  gene. The fusion gene was inserted into pUCN between the BamHI and SacI sites to obtain pUCNOA. pUCNOA was double digested with HindIII and SacI. The 2202 bp exogenous fragment was collected on agarose gel, and inserted between the and SacI sites of plant binary expression vector pBI121, obtaining the plant expression vector pBINOA of the invention. The expression cassette of pBINOA is the Ole/apoA-I M  fusion gene driven by  Brassica napus  oleosin promoter. The structure of pBINOA is shown in  FIG. 1 . 1:  Brassica napus  oleosin gene promoter; 2:  Arachis hypogaea  oleosin gene; 3: thrombin cleavage site; 4: apolipoprotein A-I- Milano  gene. By sequencing of pBINOA, the sequence of the expression cassette is obtained as shown in SEQ ID NO: 15, with the length of 2202 bp. 
       Example 2 
     Construction of Seed-Specific Plant Expression Vector pBINOA 
       [0068]    The construction of the plant expression vector pBINOA is shown in  FIG. 2 . The specific procedures are provided as follows. 
         [0000]    Cloning of  Brassica napus  oleosin gene promoter:  Brassica napus  is an important oil crop. The oil content is up to 42˜45%. The 20 kD oleosin in  Brassica napus  oil body is 10 times the amount of 24 kD oleosin. Forward primer pBINOA-1: CCC  AAG CTT  TTC AAC GTG GTC GGA TCA TGA CG (SEQ ID NO:1) and reverse primer pBINOA-2: CGC  GGA TCC  GAA TTG AGA GAG ATC GAA GAG (SEQ ID NO:2) for the PCR amplification of  Brassica napus  20 kD oleosin gene promoter were designed according to the nucleotide sequence of  Brassica napus  oleosin gene promoter (Genbank No. AF134411) in which HindIII and BamHI cleavage sites were introduced (the underlined section). Using the genome DNA of  Brassica napus  Qingyou 14 variety as the template and pBINOA-1 and pBINOA-2 as primers,  Brassica napus  oleosin gene promoter was amplified by PCR with the following conditions: 94° C. 1 min, 63-73° C. 1 min, and 68° C. 1 min, and 10 min of extension at 68° C. after 30 cycles. The amplification product was recovered by agarose gel electrophoresis, double digested with HindIII and BamHI, and connected to pUC19 digested with HindIII and BamHI. The ligation product was mixed with 2004 of DH5α competent cell (purchased from Tiangen Biotech (Beijing) Co., Ltd.), and then subjected to ice bath for 30 min, heat shock for 1.5 min at 42° C., and ice bath for 3 min. 8004 LB culture medium was added and cultured for 45 min at 37° C. Aliquots of the transformation reaction was plated on LB agar containing 50 μg/mL ampicillin and incubated overnight at 37° C. The transformants were screened by PCR using pBINOA-1 and pBINOA-2 as primers. PCR conditions were 94° C. 1 min, 60-73° C. 1 min, 72° C. 1 min, and 10 min of extension at 72° C. after 30 cycles. The PCR product was subjected to electrophoresis with agarose gel for verification. The positive transformant was named as pUCN. The positive transformant was shaken in liquid culture medium. Plasmid was extracted through alkaline lysis. The plasmid was subjected to single enzyme digestion identification with HindIII and double enzyme digestion identification with HindIII and BamHI. The results displayed by agarose gel electrophoresis are shown in  FIG. 3 . M: DNA Molecular Weight Marker λDNA/EcoT14I; L1: product of restriction enzyme digestion of pUCN plasmid with HindIII as 3565 bp fragment; L2: products of double digestions of pUCN plasmid with HindIII and BamHI, as the vector fragment of 2662 bp and the promoter of 903 bp; and L3: promoter of 903 bp obtained from PCR detection of pUCN plasmid. pUCN is sequences according to the following procedures: (1) Using pUCN as template, conduct PCR reaction with pUC19 common sequencing primer to obtain PCR product; (2) purify PCR product to remove enzyme, florescent dye, primer, and other ions; (3) use 3730 sequencer (ABI Ltd.) to sequence the purified PCR product after degeneration and ice bath; (4) automatically analyze and print out colored sequencing map and DNA sequence by the machine. The length of the exogenous fragment in pUCN is 903 bp. The sequence is shown in SEQ ID NO:3. The molecular weight is 556.7 kDa. The enzyme digestion results and sequencing results suggest that,  Brassica napus  oleosin gene promoter was successfully cloned into pUC19. 
         [0069]    Artificial synthesis of apolipoprotein A-I- Milano  gene: Based on apolipoprotein A-I gene sequence (SEQ ID NO:4, NM000039) (amino acid sequence shown in SEQ ID NO:5) and the codon usage of  Helianthus annuus  (http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=4232), as well as the GC content in  Helianthus annuus  (genome, apolipoprotein A-I- Milano  gene is redesigned and synthesized. Residue C at position 517 was mutated into T, and at the 5′ end of the gene a thrombin cleavage site was added, with the nucleotide sequence shown in SEQ ID NO:6 (CTGGTCCCAA GGGGTAGC) and the amino acid sequence shown in SEQ ID NO:7 (L V P R G S). The molecular weight of the synthesized apolipoprotein A-I- Milano  gene was 462.4 kDa, and the sequence is shown in SEQ ID NO:8. The encoded protein is composed of 249 amino acid residues and the molecular weight is 28.585 kDa. 
         [0070]    Amplification of Ole/apoA-I M  fusion protein gene: Two pairs of specific primers (pBINOA-3/pBINOA-4 and pBINOA-5/pBINOA-6, wherein the sequences are SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12 respectively) were designed according to the sequence of  Arachis hypogaea  oleosin gene (Genbank No. AF325917) and the sequence of apolipoprotein A-I- Milano  gene (SEQ ID NO:8). pBINOA-3 and pBINOA-6 were provided with BamHI and SacI restriction sites (the underlined section) respectively. Moreover, Kozak sequence (the bolded part in the sequence, to improve the transcription and expression efficiencies) is positioned near the initiator codon of oleosin gene in pBINOA-3 primer. pBINOA-4 and pBINOA-5 were reverse complementary sequences. 
         [0000]    
       
         
               
             
           
               
                 pBINOA-3: 
               
               
                 CGC  GGA TCC   AGC AAA GCC GCC ACC  ATG GCT ACT  
               
               
                   
               
               
                 GCT ACT GAT CG 
               
               
                   
               
               
                 pBIN0A-4: 
               
               
                 GCT ACC CCT TGG GAC CAG TGA TGA TGA CCT CTT  
               
               
                   
               
               
                 AAC 
               
               
                   
               
               
                 pBINOA-5: 
               
               
                 GTT AAG AGG TCA TCA TCA CTG GTC CCA AGG GGT  
               
               
                   
               
               
                 AGC 
               
               
                   
               
               
                 pBINOA-6: 
               
               
                 C  GAG CTC  TTA TTG TGT GTT AAG TTT CTT TG 
               
             
          
         
       
     
         [0071]    Using pBINOA-3/pBINOA-4 as the primer,  Arachis hypogaea  (variety Jihua 4) genome DNA as template, the  Arachis hypogaea  oleosin gene lacking the terminate codon was amplified. PCR conditions are 94° C. 1 min, 50-55° C. 1 min, 68° C. 1 min, and 10 min of extending at 68° C. after 30 cycles. Using pBINOA-5/pBINOA-6 as the primer, the optimized apolipoprotein A-I- Milano  as template, the apolipoprotein A-I- Milano  gene was amplified. PCR conditions are 94° C. 1 min, 63-73° C. 1 min, 68° C. 1 min, and 10 min of extending at 68° C. after 30 cycles. The two PCR amplification products were recovered by agarose gel electrophoresis, and mixed at the molar ratio of 1:1 to serve as template. pBINOA-3/pBINOA-6 were used as primer for overlapping PCR. PCR conditions are 94° C. 1 min, 50-55° C. 1 min, 68° C. 2 min, and 10 min of extending at 68° C. after 30 cycles. Ole/apoA-I M  fusion gene was obtained through agarose gel electrophoresis of the amplification product. The construction of Ole/apoA-I M  fusion gene is shown in  FIG. 4 . M: DNA molecular weight marker DL2000; L1: the 528 bp fragment of  Arachis hypogaea  oleosin gene lacking the termination codon, amplified with pBINOA-3/pBINOA-4 as the primer and  Arachis hypogaea  (variety Jihua 4) genome DNA as the template: L2: the 750 bp apolipoprotein A-I- Milano  gene amplified with pBINOA-5/pBINOA-6 as the primer and the optimized apolipoprotein A-I- Milano  gene as the template (the nucleotide sequence containing thrombin cleavage site); L3: Ole/apoA-I M  fusion gene obtained by overlapping PCR with pBINOA-3/pBINOA-6 as the primer. The Ole/apoA-I M  fusion gene was sequenced, and the results indicated that the sequence of Ole/apoA-I M  fusion gene was as shown in SEQ ID NO:13. The length is 1278 bp, and the molecular weight is 787.9 kDa. The deduced amino acid sequence is shown in SEQ ID NO:14, comprising 425 amino acid residues. The molecular weight is 46.994 kDa. The construction results and sequencing results of oleosin-apoA-I M  fusion gene showed that, we had already obtained ole/apoA-I M  fusion gene. 
         [0000]    Construction of intermediate vector pUCNOA: The ole/apoA-I M  fusion gene was BamHI and SacI double digested and ligated with pUCN which was double digested in the same way. The ligation product was mixed with 200 μL DH5α competent cell (purchased from Tiangen Biotech (Beijing) Co., Ltd.), and subjected to ice bath for 30 min, heat shock for 1.5 min at 42° C., and ice batch for 3 min. 8004 LB culture medium was added and grown at 37° C. for 45 min. LB agar plate containing 100 μg/mL ampicillin was innoculated and incubated at 37° C. overnight. The transformants were selected by PCR using pBINOA-3 and pBINOA-6 as primers. PCR conditions were 94° C. 1 min, 60-73° C. 1 min, 72° C. 1.5 min, and extension of 10 min at 72° C. after 30 cycles. The PCR product was run on agarose gel. The positive transformant was named as pUCNOA and was shaken in liquid medium. Plasmid was extracted by alkaline lysis. The plasmid was identified by HindIII single digestion identification, HindIII and BamHI double digestion identification, and BamHI and SacI double digestion identification. The identification results of agarose gel electrophoresis are shown in  FIG. 5 . M: DNA molecular weight marker λDNA/EcoT14I; L1: fragment of 4849 bp obtained by HindIII single digestion of pUCNOA plasmid; L2: vector fragment of 2647 bp and exogenous fragment of 2202 bp (containing  Brassica napus  oleosin gene promoter and ole/apoA-I M  fusion gene) obtained by HindIII and SacI double digestion of pUCNOA plasmid; L3: vector fragment of 3571 bp and exogenous fragment of 1278 bp (ole/apoA-I M  fusion gene) obtained by BamHI and SacI double digestion of pUCNOA plasmid. The pUCNOA plasmid was sequenced, and the sequencing results are shown in SEQ ID NO:15. The total length is 2202 bp and the molecular weight is 1357.5 kDa, including  Brassica napus  oleosin gene promoter and ole/apoA-I M  fusion gene. The enzyme digestion results (as shown in  FIG. 5 ) and sequencing results (as shown in Sequence List) (SEQ ID NO:15) indicated that, the expression cassette of  Brassica napus  oleosin gene promoter-driven  Arachis hypogaea  oleosin gene-apolipoprotein A-I- Milano  fusion gene was obtained and the said expression cassette was successfully cloned into the vector pUC19. 
         [0072]    Construction of seed-specific plant expression vector pBINOA: DNA of pUCNOA plasmid was extracted by alkaline lysis, and cleaved with HindIII and SacI. The exogenous fragment of 2202 bp was recovered by agarose gel electrophoresis and ligated to pBI121 cleaved with HindIII and SacI. The ligation product was mixed with 200 μL DH5α competent cell (purchased from Tiangen Biotech (Beijing) Co., Ltd.), and subjected to ice bath for 30 min, heat shock at 42° C. for 1.5 min, and ice bath for 3 min. 800 μL LB culture medium was added and cultivated for 45 min. LB plate containing 100 μg/mL kanamycin was plated and cultivated at 37° C. overnight. Transformants are screened by PCR using pBINOA-1 and pBINOA-6 as primers. PCR conditions were 94° C. 1 min, 60-73° C. 1 min, 72° C. 2 min, and extension or 10 min at 72° C. after 30 cycles. The PCR product were screened through agarouse gel electrophoresis. The positive transformant was designated as pBINOA. The positive transformant was cultured in liquid while shaking. Plasmid was extracted with alkaline lysis, and subjected to HindIII single digestion identification and HindIII and SacI double digestion. The identification results of agarose gel electrophoresis are shown in  FIG. 6 . M: DNA molecular weight marker λDNA/EcoT14I; L1: fragment of 14205 bp, the product of HindIII digestion of pBINOA plasmid; L2: vector fragment of 12003 bp and exogenous fragment of 2202 bp (including  Brassica napus  oleosin gene promoter and ole/apoA-I M  fusion gene), the products of HindIII and SacI double digestion of pBINOA plasmid. The pBINOA plasmid was sequenced, and the sequencing result is as shown in SEQ ID NO:15. The full-length nucleotide sequence of the vector is shown as SEQ ID NO:16. The entire expression cassette is 2202 bp long. The molecular weight is 1357.5 kDa, including  Brassica napus  oleosin gene promoter and ole/apoA-I M  fusion gene.  Brassica napus  oleosin gene promoter is a strong seed-specific promoter, and drives the specific expression of apolipoprotein A-I- Milano  in oil body as fusion with  Arachis hypogaea  oleosin in the transgenic plant.  Arachis hypogaea  oleosin carrying with apolipoprotein A-I- Milano  is anchored on oil body surface. Utilizing the hydrophobic/lipophilic characteristics of oil body, the transgenic plant seeds were ground and extracted, centrifuged, and the upper oil phase recovered, thereby separating the protein from other components in the cell. More than 90% of the seed protein was removed. Thrombin recognition site was positioned between  Arachis hypogaea  oleosin and apolipoprotein A-I- Milano  to release apolipoprotein A-I- Milano  from oil body. 
       Example 3 
     Production of Apolipoprotein A-I- Milano  (AIM) with the Vector 
       [0073]    3.1 Introduce the Seed-Specific Expression Vector Constructed Above into the Explants of the Receptor Plant; 
         [0074]    3.1.1 Preparation of the Competent  Agrobacterium  Cells 
         [0075]    (1) Transfer  Agrobacterium tumefacien  LBA4404 single bacterium into 3 mL YEB medium (containing streptomycin Sm 125 μg/mL), and grow the cells at 28° C. overnight; 
         [0076]    (2) Transfer 5004 overnight culture into 50 mL YEB (Sm 125 μg/mL) medium, and grow the cells at 28° C. until OD 600  is 0.5; 
         [0077]    (3) 5,000 rpm, centrifuge for 5 min; 
         [0078]    (4) Resuspend  Agrobacterium  cells in 10 mL 0.15M NaCl solution, 5,000 rpm, and centrifuge for 5 min; 
         [0079]    (5) Resuspend  Agrobacterium  cells in 1 mL precooled 20 mM CaCl 2  for ice bath and use within 24 h, or dispense aliquots (200 μl) of the suspensions into tube and quick freeze for 1 min in liquid nitrogen, and preserve at −70° C. for later use. 
         [0080]    3.1.2. Transformation of  Agrobacterium  Competent Cells with Seed-Specific Plant Expression Vector 
         [0081]    1 μg thus constructed plasmid DNA was added to 2004 competent cells, and stored in liquid nitrogen for 1 min, in water bath at 37° C. for 5 min. Then 1 mL YEB medium was added, cultivated in liquid medium at 28° C. while slowly shaking for 4 h; and centrifuged at 1,000 rpm for 30 sec. The supernatant was discarded and 0.1 mL YEB medium was added for resuspension. Aliquots of the transformation reaction were plated on YEB agar plate containing 100 μg/mL Kan and 124 μg/mL Sm, and incubated at 28° C. for approximately 48 h. 
       Identification of Positive Clone 
       [0082]    Single colony was picked into YEB medium (containing 100 μg/mL Kan and 125 μg/mL Sm), and cultivated in liquid medium at 28° C. overnight. Small amount of plasmid DNA was extracted with alkaline lysis. Using the plasmid DNA as template and pBINOA-1 and pBINOA-6 as primers, PCR amplification identification was carried out under the following conditions: 94° C. 1 min, 60-73° C. 1 min, 72° C. 2 min, and extension of 10 min at 72° C. after 30 cycles. Positive transformants were obtained after agar gel electrophoresis of PCR product. 
       Preparation of  Agrobacterium  Suspension Used for Oil Sunflower Transformation 
       [0083]    5 mL YEB medium containing 100 μg/mL Kan and 125 μg/mL Sm was inoculated with a single colony of transformed  Agrobacterium . The culture was grown overnight with shaking. 100-200 mL YEB liquid medium containing 100 μg/mL Kan and 125 μg/mL Sm was inoculated with 1 mL culture. The culture was grown at 28° C. with vigorous shaking until OD 600  is 0.4˜0.8, and centrifuged at 3500 rpm for 10 min to recover cells. The pellet was resuspended with MS (free of plant growth regulators or antibiotics) to make OD 600  at approximately 0.6 for transformation. 
         [0084]    3.1.3 Genetic Transformation of Oil Sunflower Explants Mediated by  Agrobacterium    
         [0085]    The explants, in the forms of shoot apexes excised from sterile seedlings, cotyledon, cotyledonary node or seedlings with one cotyledon detached, of the seedling of oil sunflower seeds sprouting for 3˜4 d were immersed in said  Agrobacterium  suspension for 6˜8 min and transferred to MS solid medium for culture for 3 d (at 25° C., in dark). The seedlings with one cotyledon detached is preferred. 
         [0086]    3.2 Cultivation of the Above Receptor Plant Materials into Complete Plant to Obtain Seeds for the Detection of Target Gene and Protein 
         [0087]    3.2.1 Cultivate the Receptor Plant Materials into Complete Plant and Obtain Seeds 
         [0088]    The transformed explants were transferred to MS agar medium containing 300 mg/L cephalosporin for approximately 7 d, then transferred to MS resistance screening medium (containing 300 mg/L cephalosporin and 70 mg/L kanamycin) for selective culture. The medium was exchanged every 15˜20 d. Resistance buds were obtained after three rounds of screening. 2˜3 cm resistance buds were transferred to rooting medium MS2 (MS+IBA0.1 mg/L+Kan 70 mg/L+cef 300 mg/L) and transplanted after rootage of resistance seedling into greenhouse for vermiculite and Nutritional soil mixture culture until maturity, seeds harvested. 
         [0089]    3.2.2 Target Gene and Protein Detection 
         [0090]    PCR detection was performed on apolipoprotein A-I- Milano  gene during the Seedling Stage. Western blotting detection was performed on  Arachis hypogaea  oleosin and apolipoprotein A-I- Milano  fusion protein after harvesting kernels. 
       PCR Detection and PCR-Southern Blotting Detection of Transgenic Oil Sunflower Seedling 
       [0091]    SDS method was adopted to extract the genome DNA of the young leaves of resistant oil sunflower seedling as the template. PCR amplification was carried out with two pairs of primers nptIIF/nptIIR and pBINOA-5/pBINOA-6. The sequences of the premiers are nptIIF: ATG AAC TGC AGG ACG AGG (SEQ ID NO:17) and GCG ATA CCG TAA AGC ACG (SEQ ID NO:18) respectively. The PCR condition of nptIIF/nptIIR and pBINOA-5/pBINOA-6 includes 94° C. for 1 min, 60° C. for 1 mm, 72° C. for 1 min, and final extension for 10 min at 72° C. after 30 cycles. As anticipated, fragments of 567 bp (partial nptII gene) and apoA-I M  gene fragment of 750 bp were amplified respectively. The results are shown in  FIG. 7  and  FIG. 8 . In  FIG. 7 , M: DNA molecular weight marker DL2000; L1-L4: the fragment of 567 bp amplified with nptIIF/nptIIR as the primer and the genome DNA extracted from the kanamycin-resistant oil sunflower as the template, i.e., positive plants; L5: use non-resistant oil sunflower as control. In  FIG. 8 , M: DNA molecular weight marker DL2000; L1-L4: the fragment of 750 bp amplified with pBINOA-5/pBINOA-6 as the primer and the genome DNA extracted from the kanamycin-resistant oil sunflower as the template, i.e., positive plant; L5: use non-resistant oil sunflower as control. 
       PCR-Southern Blotting Detection 
       [0000]    
       
         1) Genomic DNA of the young leave of the transgenic oil sunflowers, in which both nptII and apoA-I M  are positive, was extracted with SDS method. PCR amplification was performed on the genome DNA with pBINOA-1/pBINOA-6 as the primer. The PCR reaction condition includes 30 cycles of 94° C. for 1 min, 60° C. for 1 min, and 72° C. for 2.5 min; and final extension at 72° C. for 10 min. 
         2) The DNA was transferred from agarose gel to a nylon membrane, denatured and neutralized after electrophoresis, and subjected to semi-dry blotting. The membrane was dried and baked for 1.2 hr at 80° C. in a vacuum oven. 
         3) DNA probe marking 
       
     
         [0095]    The pBINOA plasmid DNA digested with BamH□ and Sac□ was recovered. 3 μg DNA was used for labeling.
   4) Hybridization   
 
         [0097]    The membrane was pre-hybridized at 63° C. for 30 min and hybridized at 63° C. overnight, washed twice with 2×SSC, 0.1% SDS, and then washed twice with 0.5×SSC, 0.1% SDS preheated to 65° C. at 63° C.
   5) Detection   
 
         [0099]    The hybridized and washed membrane was briefly rinsed once with washing buffer, incubated in 100 ml Blocking solution for 30 min, incubated for 30 min in 20 ml Antibody solution, Washed 2×15 min in 100 ml Washing buffer, and equilibrated for 2-5 min in 20 ml Detection buffer. The membrane was placed in a hybridization bag (with DNA side facing up) and 1 ml CSPD added. The membrane was incubated for 10 min at 37° C. to enhance the luminescent reaction, and exposed to X-ray film at room temperature. The results are shown in  FIG. 9 . M: DNA molecular weight marker λ DNA/EcoT14I; L1-L4: the Southern blotting results of the product amplified with the positive plant genome (detected as positive by PCR) as the template and pBINOA-1/pBINOA-6 as the primer. The hybridization signal was displayed at the place of 2.2 kb as expected, suggesting the integration of ole/-apoA-I M  fusion gene into oil sunflower genome; L5: control of non-transgenic oil sunflower. 
         [0100]    Western Blotting Detection of  Arachis hypogaea  Oleosin and Apolipoprotein A-I- Milano  Fusion Protein in Transgenic Oil Sunflower Seeds 
         [0101]    Transgenic oil sunflower seeds were ground in five volumes of grinding buffer (50 mM Tris-HCl pH 7.5, 0.4 M sucrose, 0.5M NaCl), centrifugated 10×g for 30 min, and separated into three parts. The oil phase was collected and resuspend in one volume of grinding buffer and mixed even. Five volumes of precooled 50 mM Tris-HCl pH 7.5 buffer was added, centrifugated 10×g for 30 min, and the oil phase collected. The above processes were repeated for two times to further remove the remaining water-soluble ingredients and insoluble ingredients, obtaining pure oil body (the ingredients of oil body include: neutral lipids, phosphatides, and oleosin). To the oil body was added 2V of diethyl ether and centrifugated. The neutral lipids were in the upper diethyl ether layer and phosphatides were left in the lower water phase. The intermediate protein layer was collected and suspended in 0.1M sucrose buffer. Chloroform methanol (2:1) mixture was added and extracted twice. The intermediate protein layer was collected, extracted with diethyl ether once and dissolved in sterile water. SDS polyacrylamide gel electrophoresis was performed, and then Western blotting analysis was performed using polyclonal goat anti-rabbit apolipoprotein A-I after transmembrane. The results are shown in  FIG. 10 . M: protein molecular weight standard; L1 and L2: oil protein extracted from transgenic oil sunflower seeds, expression of apolipoprotein A-I- Milano  is shown. A fusion protein of molecular mass of approximately 48 kDa was recognized, consistent with the anticipated result ( Arachis hypogaea  oleosin 18.4 kDa, thrombin cleavage site 0.6 kDa, and apolipoprotein A-I- Milano  28.9 kDa). The fusion protein accounts for 1.1% of the total seed protein, exceeding the minimum commercialization requirement (1%) of recombinant medical protein in plant. Therefore, it is feasible and applicable to make use of plant oil body expression system to achieve the industrial production of apolipoprotein A-I- Milano . 
         [0102]    3.3 Obtain Apolipoprotein A-I- Milano  from the Seeds by Separation and Purification. 
         [0103]    Step 1: 
         [0104]    Separate Oil Body from Other Components in Seeds 
         [0105]    The kernal was ground in five volumes of grinding buffer (50 mM Tris-HCl pH 7.5, 0.4M sucrose, 0.5 M NaCl), centrifuged at 10×g for 30 min, and divided into three parts. The bottom part is insoluble precipitation (hull, fiber materials, insoluble sugar, protein and other insoluble dirt); the middle layer is aqueous phase, containing soluble cellular constituents (storage protein); the upper layer is the oil body and the associated oil body protein. 
         [0106]    Step 2: 
         [0107]    Wash the Oil Body 
         [0108]    The oil phase obtained from Step 1 was resuspended in the same volume of grinding butter and mixed even. Five volumes of precooled 50 m MTris-HCl pH 7.5 buffer are added and centrifuged at 10×g for 30 min. The oil phase was collected. The above processes were repeated twice to further remove the residual water-soluble ingredients and insoluble ingredients. The washed oil body was resuspended in precooled 50 mM Tris-HCl pH 7.5 of equivalent volume. The resulting oil body was substantially pure oil body, and he only protein left was oil body protein. 
         [0109]    Step 3: 
         [0110]    Release Apolipoprotein A-I- Milano  Protein by Restrictive Digestion 
         [0111]    The oil body was washed with thrombin digestion buffer (20 m M Tris-HCl pH8.4, 150 m M NaCl, and 2.5 m M CaCl 2 ) for two times. Appropriate amount of thrombin was added, stored at 37° C. overnight, and centrifuged. Apolipoprotein protein exists in the aqueous phase. 
         [0112]    Step 4: 
         [0113]    Purify Apolipoprotein A-I- Milano  Protein with High Performance Liquid Chromatography (HPLC) 
         [0114]    Reversed-phase chromatography C4 column (5μ, 0.24*25 cm) was used, at the ultraviolet wavelength of 214 nm. The column was equilibrated with 2 mL/min buffer A (10% acetonitrile, 0.1% trifluoroacetic acid), loaded with the aqueous phase obtained in the last step, and applied linear gradient elution of 0-60% buffer B (95% acetonitrile, 0.1% trifluoroacetic acid). Pure apolipoprotein A-I- Milano  protein was obtained with the purity above 99.5%. 
       Example 4 
     Comparison Between Oil Sunflower and  Carthamus Tinctorius  as Bioreactor for the Production of Apolipoprotein A-I- Milano  (AIM) 
       [0115]    The same amount (280 mg) of trans-apolipoprotein A-I- Milano  gene oil sunflower seed and  carthamus tinctorius  seed were used to obtain apolipoprotein A-I- Milano  protein by separation and purification according to Example 3. The loading quantity was one-tenth of the total quantity obtained. Western blotting detection was performed, and the results are shown in  FIG. 11 . M: protein molecular weight standard; L1: apolipoprotein A-I- Milano  purified from transgenic  carthamus  28.9 kDa as expected, with the amount of 50 ng; L2: the apolipoprotein A-I- Milano  purified from transgenic oil sunflower, 28.9 kDa as expected, with the amount of 80 ng. It can be calculated that 1 kg of transgenic oil sunflower seed can produce 2.85 g of apolipoprotein A-I- Milano , while under the same condition, 1 kg transgenic  carthamus tinctorius  seed can produce 1.78 g of apolipoprotein A-I- Milano . Moreover, the yield per mu of oil sunflower is approximately 250 kg while that of  carthamus tinctorius  is approximately 200 kg. Therefore, oil sunflower is superior to  carthamus tinctorius  in terms of the yield of apolipoprotein A-I- Milano  protein per seed weight or per plant area.