# Patent

## Title
Cell line expressing mutated human tissue-type plasminogen activator, the constructing strategy thereof and method of preparing expressed protein

## Abstract

The invention involves a cell line which can express human mutated tissue-type plasminogen activator (TNK-TPA), and its preparation methods. The collection number of cell lines in this invention is CCTCC C200006. We first used a DNA fragment from the short arms of human group D or group G chromosomes or its homolog to construct a recombinant human source gene vector-TNK-TPA (collection number is CCTC m200032); TNK-TPA gene is then incorporated into the target site in the nucleolus organizing region (NOR) of human group D or G chromosomes in host cell line HT1080; of which the recombinant cell line is attained after screening. The recombinant cell line can be used for manufacturing protein.

## Background
This application is the national phase under 35 U.S.C. § 371 of PCT International Application No. PCT/CN01/00127 which has an International filing date of Feb. 16, 2001, which designated the United States of America.
SUMMARY OF THE INVENTION
The invention relates to a cell line specifically expressing mutated human tissue-type plasminogen activator (TNK-TPA), the construction strategy for said cell line and application of the cell line in manufacturing recombinant human tissue-type plasminogen activator
BACKGROUND OF THE INVENTION
Human tissue-type plasminogen activator (t-PA) can dissolve local thrombi in blood vessels and recanalize a blocked vessel by activating plasminogen. At present, Genentech (USA) is the only entity that can produce recombinant human t-PA(rt-PA) at a large scale by means of genetic engineering technology and has developed successfully a rt-PA medicine. The first generation of rt-PA made in this company has become the treatment of choice for thrombotic diseases, but it has some shortcomings First, its price is rather high at $1375 per vial on the international market. Second, its function has some weak points: 1)short half-life, just 3 to 5 minutes; 2)low specific affinity with fibrin; 3) activity and efficacy should be further improved. A much higher dose should be given because of the short half-life. Low specificity and activity give rise to two results. On one hand, there is still 10-20% patient's thrombi that cannot be completely dissolved by rt-PA. On the other hand, it results in fibrinolysis over all the body and affects the normal blood clotting mechanism so as to cause severe complications such as bleeding, especially in the skull and digestive tract.
For the above reasons, some other companies have developed the second generation of rt-PA since the rt-PA was used in the clinical. They modified the rt-PA gene in order to eliminate or improve upon the above weak points. TNK-TPA is a kind of mutated form of rt-PA which is universally researched. For example, Keyt BA, Paoni NF, Refino rt CJ, et al proclaimed “a Faster-acting And More Potent Form of Tissue Plasminogen Activator” ((Proc Natl Acad Sci USA) 1997 Apr. 26; 91(9): 3670-4). It was constructed on the basis of three sites of the t-PA gene by site-directed mutation. Two sites are located in the Kringle function domain, T103N and Q117N; one site is located in the protease domain, “KHRR296-299AAAA” (K296A, H297A, R298A, R299A). T103N adds a glycosylation site which increases specificity of complexing of TNK-tPA and fibrin and decreases its cleanup rate; Q117N destroys combination of K1 region and mannose and the action domain of Kupffer cells in liver, and prolongs half-life; KHRR296-299AAAA destroys the functional region of PAI-1 and prolongs its half-life, too. As for the production of TNK-TPA, it is universal to adopt an ordinary eukaryotic gene expression vector, using CHO cells as the host cell. TNK-TPA integrates randomly into the expression system of the host cell genome. For example, Wu Benchuan, et al, announced the above expression system and preparation method of expressed protein in the article “Purification and identification of recombinant tissue-type plasminogen activator” published in <Development of Biochemistry and Biophysics> (1997, 24 (1):71-75).
DETAILED DESCRIPTION OF THE INVENTION
The inventor discovered two families carrying an extra bi-satellite microchromosome (BM) in these two families with 2 and 3 generations, the BM does no harm to human body. The families show a normal phenotype. Seventeen similar families are reported in the world, and nobody conceived of assembling human gene vector using the small chromosome elements. The applicant put forward the structure Using the FISH technique, it was found that the bi-satellite microchromosomes come from the short arms of human group D or G chromosomes 13, 14, 15, 21 and 22. The short arms of human group D or G chromosomes are nucleolus organizing regions abundant in ribosomal DNA (rDNA). Different polymorphisms with different length exist in these regions in human chromosomes, and the genes in these regions can be actively transcribed in the cell metaphase. Thus it was inferred that specific DNA fragments can be used as a targeting sequence if they were isolated from BM, and the gene of interest can be transferred into the nucleolus organizing region in the short arms of human group D or G chromosomes. There the gene should be relatively highly, stably and harmlessly expressed The following example can prove this strongly.
The inventor at first constructed a specific pUC19 library of the microchromosomes by a micro-dissection method and obtained a single copy fragment by screening the library The fragment was proved by the FISH technique to come from the short arms of human group D or group G chromosomes and the single copy was used as probe to screen a human PAC genomic library. Then, a 120 kb DNA fragment which comes from BM and short arms of human group D or group G chromosomes was confirmed by the FISH technique (FIG. 1). No gene concerned with important physiological function is found by analyzing sequence of this BM specific fragment (BMSF), so it is safe to take it as a target The applicant further used a small fragment from the BMSF as a targeting sequence to construct a gene vector.
Based on above evidence, a gene vector using a DNA sequence without vital physiological function-related gene or a DNA sequence with identity to short arms of human group D or G chromosomes as a targeting sequence can site-directly introduce a desired gene into the short arms of human group D or group G chromosomes and so the cell line according to the invention can then be obtained.
Different vectors can be constructed on the basis of the targeting sequence according to the present technique, and then one can obtain a recombinant vector-TNK-TPA by ligating a TNK-TPA gene into the constructed vector The methods used for constructing the targeting vector and the TNK-TPA vector are common in the art. Using the recombinant above, the desired gene TNK-TPA can be also transferred into a host cell by routine method.
The example of the invention gives in detail the identification of the 120 kb specific DNA sequence from short arms of human group D or group G chromosomes, and the construction of the vector using a 3.8 kb fragment from the 120 kb BMSF as a targeting ing sequence. The full sequence of the vector is given in SEQ ID NO: 1. Then a TNK-TPA gene as a desired gene is ligated into the vector and the recombinant vector-TNK-TPA is achieved. The inserting site of TNK-TPA gene is at nucleotide 5910 of the vector. The bacterial strain containing the recombinant vector-TNK-TPA is preserved in the China Typical Culture Collection Center (Wuhan University, China. Wuhan 430072) on Sep. 29, 2000. The collection number is CCTCCM200032. The depositor names it Escherichia coli JM109/JH-5/pNS-TPA.
According to the above methods, a novel cell line with the desired gene TNK-TPA integrated in the short arms of human group D or group G chromosomes of an E. coli HT1080 cell. The cell line was accepted by China Typical Culture Collection Center (Wuhan University, China. Wuhan 430072) on Aug. 18, 2000. The collection number is CCTCC C200006. The cell line is a human fibrosarcoma cell line, which is human gene-transformed cell. The depositor names it human fibrosarcoma cell line /JH-2/TPA. The preserved cell line is a pure cultivative cell line. The cells may be cultured in 10% calf serum in DMEM, pH 7.0-7.4, at 37° C.
The above cell line can substitute for transformed CHO transformed cells to prepare TNK-TPA.
The purified protein expressed by the cell line of the invention has been confirmed to be the desired protein by Western blotting and amino acid sequence analysis.
The invention provides a novel cell line by site-directedly transferring a recombinant human-source gene vector/TNK-TPA cDNA into D group and/or G group chromosomes of a human HT1080 cell and so provides a method to produce TNK-TPA Compared with the existing technology, the invention dramatically decreases cost of TNK-TPA because of its efficient and stable expression.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows FISH mapping of 120 kb BMSF fragment cloned in PAC.
FIG. 2 is a circular map of the gene vector described in the invention (length of gene vector sequence: 1162 bp); pGEM-7 (8267-11162): vector replication component and prokaryote screening system; TK(1-2840): eukaryote negative screen gene, using TK promoter and TK polyA signal; Neo(4342—5910): eukaryote positive screen gene, using sv40 promoter and sv40 polyA signal; GLS (2841—4341,5911—8267): leading sequence for gene targeting; Cloning site (5910): insert-site of desired gene
FIG. 3 is FISH mapping of targeted TNK-TPA gene in positively a transformed cell colony. This revealed that the TNK-TPA gene has been site-directedly transferred into the short arms of human group D or group G chromosomes by the vector.
FIG. 4 is Western blotting result of purified TNK-TPA protein, 1 ̃4 are purified TNK-TPA protein, “−” denotes negative control.



EXAMPLE 1
Preparation of a Gene Targeting Sequence of the Invention
1 Isolation of PAC Clone with Gene Targeting Sequence

    1.1 Construct BM specific pUC19 library through micro-dissection, PCR, microcloning (Deng H-X, Yoshirua K, Dirks R W, et al. Hum Genet 1992,89:13.)
    1.2 Identification of BM Specific Single Copy DNA


(1) preparation of colony matrix membrane: draw 14×12 square wells on two nylon membranes, labeled as A and B, and put into two plates with solid LB, respectively, then pick white clones randomly from library plates and inoculate to the 14×12 square wells of two membranes with same coordinate, add 100 ng single copy DNA to the 13th line as positive control, and then add 100 ng gDNA to 14th line as negative control. Two plates are incubated for 10 ̃12 hours at 37° C., and membrane B is stored at 4° C. Remove membrane A from plate, and treat on a filter paper soaked by following solution: 10% SDS, 5 minutes, 0.5N NaOH/1.5M NaCl, 3 minutes, 1.5M NaCl/0.5M Tris.HCl, 3 minutes, 2×SSC/0.2M Tris.HCl, 10 minutes, 80° C. vacuum dry 2 hours, keep for use.
(2)Preparation of gDNA Probe
Sample 50 ̃75 ng gDNA, add sterile water to 11 ml, boil and denature at 100° C. for 10 minutes. The following system is used as a random primer labeling reaction:
















2 mM dNTP(dATP−)
3 μl



primer mixture
2 μl



Klenow enzyme
1 μl



α-32p-dATP
3 μl










vortex and mix up thoroughly, incubate 30 minutes at 37° C. Add 8 μl stop mixture, pass through G-50 column to purify probe, withdraw 1/10 of the mixture to conduct liquid scintillation.

(3) Hybridization: colony matrix membrane is immersed in 2×SSC 10 minutes, lightly wipe off colony fragments from the membrane surface. Pre-hybridize at 65° C. in 5 ml hybridization solution over 30 minutes. Sample probe solution to boil and denature 10 minutes at 100° C. according to the concentration of 1.2×106 cpm/ml and measure liquid scintillation value, add 5 ml fresh hybridization solution, hybridize with colony lattice membrane over 12 hours at 65° C., and wash membrane according to following conditions: 2×SSC/0.1% SDS, 10 minutes at room temperature, 2×SSC/0.1% SDS, 10 minutes at 65° C., 0.1% ×SSC/0.1% SDS, 10 minutes at 65° C., −70° C., autoradiograph. No or a weak hybridization signal is primarily regarded as single copy.
(4) Sequence analysis, Southern blotting: pick a clone from the corresponding position of membrane B, culture it in one small scale, sequence plasmid DNA extracted. The sequence of single copy DNA shows no similarity compared with the database of GenBank. At last, digest the plasmid by EcoRI enzyme and isolate the insert DNA by random primer method, label the insert by α-32p-dATP and hybridize with gDNA digested by EcoRI on a piece of nylon membrane, those showing 1 or 2 hybridization bands are single copy DNA clones.

    1.3 Identification of PAC Clone with BM Specific Sequence and Short Arms of Human Group D or Group G Chromosomes


(1) Screen Human PAC gDNA and Acquire Positive Clone
(2) Label 260 bp single copy probe P8-7 with α-32p-dATP by random primer method→purify probe by G-50 column (medium granularity)→ready for use 4° C. →immerse 7 PAC membranes in 2×SSC 10 minutes→pre-hybridize 3 hours, 55° C. →denature probe 10 minutes, 100° C. →add to 50 ml hybridization solution purchased commercially to a final concentration of 4.6×105 cpm/ml and hybridize with PAC membrane 1 hour 65° C. →wash membrane: 2×SSC, once a time 10 minutes at room temperature, 2×SSC/0.1% SDS, 10 minutes at 65° C. twice→apply to X-ray film, autoradiograph at 70° C. 12 hours→develop X-ray film→read the positive clones.
(3) Randomly pick positive clones and number from five different plates, purchase PAC clone.

    1.4 Hybridization of the DNA fragment in PAC clone with metaphase cells confirms that the insert DNA in positive PAC clone originates from short arms of human group D or group G chromosomes through FISH, as the FIG. 1 shows.


The above experimental methods refer to the Molecular Cloning written by J Sambrook et al., second Edition, Cold Spring Harbor laboratory Press, 1989.
2. Isolation of the targeting sequence. Main material: β-agarase(Bio-Labs), Not I Agarose
(1) Digest the plasmid PAC169 by Not I enzyme
(2) Isolate an around 120 kb insert DNA through PFGE.
Pulse electrophoresis conditions electrophoresis buffer: 0.5×TBE High strength Analytical
Grade Agarose: (Bio-Rad, low Melting point agarose LMP) 1%,
Switch time: from two to fifteen second, electrophoresis time: 18 h. voltage: 6v/cm, angle: 120°, temperature: 14° C.
(3) After electrophoresis, stain the gel with EB (0.2 ug/ml) thirty minutes. According to the Marker display, cut that 120 kb DNA band with sterile knife.
(4) Treat the gel cut out by β-agarase and precipitate with enthanol.
EXAMPLE 2
Preparation of Gene Vector of the Invention
1. The Construction of Gene Vector and Induction of Target Gene
1.1 Construct Gene Vector
1.1.1 Nsi I and Stu I (blunt enzyme) digest PAC DNA, run the sample on a common agarose gel, recover the 3.8 kb DNA and purify the DNA by electric elution.
1.1.2 pGEM-TK vector DNA is digested with Hind III, and then digested with Klenow further to generate the blunt ends.
1.1.3 The blunt ends of pGEM-TK/Hind III is further digested with Nsi I.
1.1.4 The purified 3.8 kb/Nsi I+Stu I digested DNA and the Nsi I digested pGEM-TK are ligated for 17 hours at 16° C.
1.1.5 The ligated product is transformed into JM109 competent bacteria, culture 19 hours at 37° C. in an ampicillin plate.
1.1.6 Randomly pick single clones and identify the positive clones with Nsi I and Nhe I double digestion.
1.1.7 The plasmid pCDN-GPR is digested with Xba I and Nhe I to acquire the XbaI+NheI digested Neo gene.
1.1.8 Construct pNS2 gene vector by ligating the XbaI+NheI digested Neo gene with the pGEM-TK-3.8 kb/Nhe I fragment.

    2.1 Induction of TNK-TPA Gene


2.1.1 Clone TNK-TPA (CDS) into pcDNA 3.1(-).
2.1.2 Design the primers TPCF and TPCR to amplify TNK-TPA gene and expression element (CMV promoter and BGH polyA signal), Avr II restrictive sites are added to the two ends of the primers.









Primer Sequence:

(SEQ ID NO:2)








TPCF:
ATGCATCCTAGGGGAGGTCGCTGAGTAGTG



       AvrII


 









(SEQ ID NO:3)








TPCR:
TGCATGCCTAGGTACCCCCTAGAGCCCAG



       AvrII






2.1.3 Digest TNK-TPA gene and expression element (CMV promoter and BGH polyA signal) with AvrII, ligate it to the pNS2 vector digested with Nhe I.
2.1.4 Transform the ligated product into recombinant JM109 E. coli to obtain a bacterial strain containing the vector-TNK-TPA (collection number is CCTCC M200032).
The above experimental methods refer to the Molecular Cloning written by J Sambrook et al., second edition, Cold Spring Harbor laboratory Press. 1989.
3 The Gene Vector Extraction

    3.1 Material


3.1.1 QIAGEN Plasmid Maxi Kit
3.1.2 Culture Medium: Liquid LB

















Trypton
5
g



Yeast extract
2.5
g



NaCl
2.5
g



Add H2O to
500
ml



Sterilize by autoclaving










3.1.3 Ampicillin: 100 mg/ml (1000×)

    3.2 Method
    
        1) Pick positive single clones and inoculate them in 3 ml LB (Amp+), incubate for 1 h at 37° C., 250 rpm.
        2) Inoculate 100 ul of primary culture in 100 ml LB (Amp+), incubate for 16 h at 37° C., 250 rpm.
        3) Harvest the bacteria by centrifugation at 6000×g for 15 minutes at 4° C.
        4) Resuspend the bacterial pellet in 10 ml of buffer P1.
        5) Add 10 ml of buffer P2, mix gently but thoroughly 6 times, and incubate at room temperature for 5 minutes.
        6) Add 10 ml of chilled buffer P3, mix gently by inverting 6 times, and incubate on ice for 20 minutes.
        7) Centrifuge at 2000×g for 30 min at 4° C.
        8) Transfer supernatant containing plasmid DNA promptly into a 40 ml high-speed centrifuge tube and centrifuge at 2000×g for 15 min at 4° C.
        9) Equilibrate a QIAGEN tip 500 by applying 10 ml buffer QBT.
        10) Transfer the supernatant to the QIAGEN-tip 500 and allow it to pass through this column.
        11) Wash the QIAGEN-tip with 30 ml buffer QC.
        12) Elute DNA with 15 ml QF and collect elution.
        13) Precipitate DNA by adding 10.7 ml (0.7 volumes) isopropylalcohol to the elution, mix up.
        14) Centrifuge immediately at 15000×g for 30 min at 4° C.
        15) Remove the supernatant, wash DNA pellet with 5 ml of 70% ethanol and centrifuge at 15000×g for 10 min.
        16) Remove the 70% ethanol, air-dry the pellet for 10 min, and dissolve the DNA in TE solution.
    
    


EXAMPLE 3
Transfer the recombinant vector-TNK-TPK acquired in the example 2 into host cell HT1080, obtain the required cell lines after screening, and express in vitro, confirms site-directed integration and high efficiency expression.
1 Material

    1.1 Cell: HT1080


Culture Medium: High Sugar DMEM+10% FBS(HT1080) EMEM+10% FBS

    1.2 Electroporation apparatus: purchased from Bio-Rad Company.


2. Methods

    1) Culture the cells in a 75 cm2 culture bottle to 70% ̃80% confluence.
    2) Harvest the cells and wash with HeBs Buffer for two times, then account the cells.
    3) Centrifuge at 15000 rpm at 4° C. for 10 min.
    4) Resuspend the cells with a reasonable volume of HeBS Buffer to make the cell density to 106 ̃107/ml.
    5) Add 0.8 ml suspension (about 10 μg DNA vector) to an 0.4 cm electroporation cuvette
    6) Electroporate the suspension, parameters: 260V, 550 uF for 11 ̃13 ms.
    7) Transfer the shocked cells to a 75 cm2 culture flask, add 14 ml culture medium with ampicillin/streptomycin, then culture in 37° C., 5% CO2 for 24 ̃48 hrs.
    8) Screen the cell with G418 (final concentration of 300 μg/ml), change medium every 2-3 days, in parallel, normal cells are used as a control.
    9) Calculate the survival clone number in transfected cells after normal cells completely die in 7-10 days, and then add G418 (150 μg/ml) to maintain surviving clones.
    10) Continue to screen the transfected cells with GCV (the ultimate concentration of 500 ng/ml)
    11) Most cells die in 7-10 days, add GCV 250 μg/ml to remaining living cells or withdraw GCV, culture the cell up to 70% ̃80% confluence, and determine the expression activity of the transfected gene.


3 Results
The TNK-TPA gene with the vector were transferred into HT1080 cells by electroporation and the positive cell lines were obtained after positive and negative screening (Collection NO. CCTCC: C200006), and confirmed to be site-directed insertion by FISH (FIG. 3). The results of assay of TNK-TPA activity are shown in Table 1.
Expression activities are: negative control HT1080 cells is 0 U/106 cells/24 hrs, the positive HT1080 cells is 408 U/106/24 hrs after transfer and 407 U/106 cells/24 hrs after 95 days. The expression of TNK-TPA is highly stable. In addition, the expressed protein of TNK-TPA is proved by Western Blotting and amino acid sequencing (FIG. 4).







TABLE 1







The results of activity assay of TNK-TPA for positive


HT1080 cells(ug/106 cells/24 hrs)












The days

The days




of transfer
T1*
of transfer
T15*
















33
408
54
188



37
396
58
204



60
411
95
114



68
430



74
430



88
441



90
440.9



95
407







*T1, T15 are two positive cell lines expressing TNK-TPA






4 The Technology of TNK-TPA Protein Preparation

    1) Culture TNK-TPA/HT1080 cell line with DMEM medium till 80% confluence, and continue to culture them with serum-free, collect the supernatant after 24 hrs which containing expressed TNK-TPA;
    2) Filtrate the supernatant with 0.22 μm filter membrane.
    3) Concentrate the filtrate 7 times with 10 KD ultrafiltration membrane.
    4) Pass through PROSEP-LYSINEII chromatography media column.
    5) Wash out the nonspecific protein absorbed on the column with 5× column volume of 0.05M PB buffer (pH 7.0).
    6) Elute TNK-TPA protein bound to the column with 0.2M Arginine/0.05M PB (PH7.5);
    7) Desalt protein solution with 3 KD ultrafiltration membrane, lyophilize it into powder dosage.

## Claims

1. A human fibrosarcoma cell line deposited as CCTCC C200006.

2. An Escherichia coli strain deposited as CCTCCM200032.