DNA CODING FOR RINDERPEST VIRUS ANTIGEN, ITS PREPARATION AND USE

Abstract: The invention provides, inter alia DNA coding for the entirety or a part of an antigen protein of the rinderpest virus; a process for the production of such DNA comprising the steps of preparing an mRNA from the rinderpest virus, preparing a cDNA library from the mRNA, selecting a cDNA coding for the target protein from the cDNA library, and cloning the selected cDNA in a cloning vector; and a process for the production of the antigen protein of the rinderpest virus comprising the steps of transfecting a vector containing the DNA coding for the target protein into animal cells, culturing the animal cells to produce the antigen protein, and recovering the target protein from the cell culture.

Description: The present invention relates, inter alia, to DNA coding for antigen proteins of the rinderpest virus, a process for the production of that DNA, and a process for the production of the antigen protein using that DNA. Rinderpest is often prevalent in many areas of the world, such as the Middle and Near East and Africa etc., and, therefore, there is intensive research into the development of vaccines for the rinderpest virus. In the development of vaccines through genetic engineering, a process wherein a gene coding for an antigen protein of the vaccine is expressed in E. coli , yeast or animal cells to obtain the antigen protein, and processes wherein a gene coding for an antigen protein is integrated into a vaccinea virus to construct a recombinant virus for the production of a live vaccine have been suggested. To carry out such process, a gene coding for an antigen protein of the rinderpest virus must be cloned. A rinderpest virus strain L that can be grown in Vero cells has been constructed (F. Kobune et al, Archives of Virology , 68 , 271-177, (1981). Also, a rinderpest virus strain L (L 13) that can be grown in Vero cells has been developed ( J. Gen. Virol . 67 , 271, 1986). The cloning of a cDNA coding for an F protein of the canine distemper virus has been disclosed by S.E.H. Russell, J. Gen. Virol . 66 , 435-441, 1985. Nevertheless, a gene coding for an antigen protein of the rinderpest virus has not hitherto been cloned. Accordingly, the present invention provides cloned genes coding for an antigen proteins of the rinderpest virus and describes process for the production thereof, and a process for the production of an antigen protein which is promising as an active ingredient for vaccines against the rinderpest virus. More particularly, the present invention provides DNA coding for the entirety or a part of an antigen protein of the rinderpest virus wherein said antigen protein is: a) a hemagglutinin protein encoded by the nucleotide sequence: or a DNA sequence hybridizable with the aforesaid sequence and encoding part at least of a rinderpest virus antigenic polypeptide or a DNA sequence as aforesaid modified by the deletion or addition of one or more codons or the modification of one or more codons but still encoding a rinderpest virus antigenic polypeptide or b) a fusion protein encoded by the nucleotide sequence: or a DNA sequence hybridizable with the aforesaid sequence and encoding part at least of a rinderpest virus antigenic polypeptide or a DNA sequence as aforesaid modified by the deletion or addition of one or more codons or the modification of one or more codons but still encoding a rinderpest virus antigenic polypeptide. The present specification also describes a process for the production of the above-mentioned DNA comprising the steps of: preparing an mRNA from the rinderpest virus; preparing a cDNA library from the mRNA; selecting a cDNA coding for the target protein from the cDNA library; and cloning the selected cDNA in a cloning vector. The present specification also describes a process for the production of the above-mentioned protein comprising the steps of: transfecting a vector containing the DNA coding for the target protein into animal cells; culturing the animal cells to produce the antigen protein; and recovering the target protein from the cell culture. In the drawings:- Figure 1 represents a restriction enzyme cleavage map of a cDNA insert RV-H in a plasmid pDH-RVH and a restriction enzyme cleavage map of a cDNA insert RV-F in a plasmid pDH-RVF; Fig. 2 represents a restriction enzyme cleavage map of a cDNA insert RV-H-2 in a plasmid pDH-RVH-2 and a restriction enzyme cleavage map of a cDNA insert RV-F-2 in a plasmid pDH-RVF-2, wherein the thick line in the RV-H-2 represents a portion corresponding to a nucleotide sequence coding for a hemagglutinin protein (H protein) of the rinderpest virus shown in Fig. 3; and, Figs. 3-1 to 3-4 represent a nucleotide sequence containing a hemagglutinin gene of the rinderpest virus, wherein, in the sequence, ATG at the 21st to 23rd position is a translation start codon and TAG at the 1848th to 1850th position is a translation stop codon, and a reading frame flanked by these codons (represented by a thick line in Fig. 2, RV-H-2) shows the codes for a hemagglutinin protein. Figs. 4-1 to 4-5 represent a nucleotide sequence containing a fusion protein gene of the rinderpest virus, wherein, in the sequence, ATG at the 587th to 589th position is a translation start codon and TAG at the 2225th to 2227th position is a translation stop codon, and a reading frame flanked by these codons (represented by a thick line in Fig. 2, RV-F-2) shows the codes for a fusion protein. A. Cloning of genes Since it is believed that proteins effective as antigens to the rinderpest virus are the hemagglutinin protein (H protein) and the fusion protein (F protein), according to the present invention, the genes coding for these proteins were cloned. (1) Extraction of mRNA The rinderpest virus L strain that can be grown in Vero cells (rinderpest virus L 13 strain) was cultured, mRNA was extracted from the culture, and the cDNA was prepared from the mRNA according to the Okayama-Berg method (Okayama and Berg, Mol. Cell Boil. , 2 , 161-170, 1983; and Mol. Cell Biol. , 3 , 280-289, 1983). More specifically, the rinderpest virus L 13 was used as the origin of the mRNA. The L 13 strain was obtained by fusing a rabbit lymphocyte infected with a rabbit-adapted passaged strain of the rinderpest virus (L-lapinized strain) and F-Vero cells, under polyethylene glycol, and passaging 15 generations. Sub-confluent F-Vero cells in eight Petri dishes each having a diameter of 6 cm were inoculated with the L 13 strain at an m.o.i. of 1. The culture was passaged on the next day, and 24 hours after the passage, 20 µg/ml of actinomycin D was added to the culture to inhibit synthesis of cellular mRNA and incubation was continued for an additional 5 hours. The recovered cells were centrifuged in 6 M of guanidinumisothiocyanate (GTC), 5 mM of sodium citrate and 0.5% of Sarkosyl - 5.7 M of CsCl₃ and 0.1 M of EDTA at 35,000 rpm (150,000 XG) for more than 12 hours, according to the guanidinum-cesium chloride method, to recover the RNA (T. Maniatis et al., Molecular Cloning , p 196, 1982). The thus-recovered RNA was the applied to an oligo (dT) cellulose column, and the adsorbed RNA was eluted by 10 mM Tris-HCl (pH 7.5) buffer containing 1 mM EDTA and 0.05% SDS to obtain a poly A (+) RNA according to a known procedure (T. Maniatis et al., Molecular Cloning , p 197, 1982). (2) Preparation of probes According to the present invention, to confirm the presence of a target mRNA in the above-prepared poly A (+) RNA, and to screen a cDNA library as described hereinafter in detail, an H gene of a subacute sclerosing panencephalitis (SSPE) virus was used as a probe (designated as H gene probe or SSPE-H-cDNA probe) to detect and screen the gene coding for the H protein (H gene) of the rinderpest virus; and the F gene of the canine distemper virus (CDV) was used as a probe (designated as F gene probe or CDV-F-cDNA probe) to detect and screen the gene coding for an F protein (F gene) of the rinderpest virus. These genes were selected as probes because the above-mentioned SSPE virus and CDV, as well as the rinderpest virus, belong to the common genus Morbillivirus and these viruses are closely related to each other, and therefore, it is considered that the nucleotide sequences thereof will have a high homology. The above-mentioned probes were prepared by obtaining an SSPE virus DNA and CDV DNA and labeling these DNA with a radioisotope ³ ⁵S (T. Maniatis et at., Molecular Cloning , p 109, 1982). More specifically, a cDNA library was made from SSPE infected cells, and SSPE H cDNA was selected by the use of measles virus H probe (Billeter et al. 1984, Virology 145 195). (DV H DNA was made according to Russel et al . (1985) (J. Gen. Virol, 66 433.). Each DNA thus-prepared was incubated with three unlabeled deoxyribonucleotides dATP, dGTP and dTTP, and a ³⁵S-labeled deoxyribonucleotide ³⁵S-dCTP-γs in the presence of E. coli DNA polymerase I in a Tris-HCl (pH 7.8) buffer. The labeled DNA was then separated from unreacted ³⁵S-dCTP-γS by a Sephadex G25 column. (3) Detection of H protein mRNA and F protein mRNA in poly A (+) RNA The poly A (+) RNA prepared as described above was denatured with glyoxal or formamide, and separated by Agarose gel electrophoresis, and the separated mRNAs were subjected to hybridization with the above-mentioned SSPE-H-cDNA probe or CDV-F-cDNA probe according to the conventional Northern hybridization method. As a result, a 1.96 kb mRNA which hybridizes with the SSPE-H-cDNA probe and a 2.2 kb mRNA which hybridizes with the CDV-F-cDNA were detected. This result suggests that the poly A (+) RNA preparation includes an mRNA coding for the H protein of the rinderpest virus and an mRNA coding for the F protein of the rinderpest virus. (4) Preparation of cDNA library A cDNA library was prepared from the above-mentioned poly A (+) RNA preparation according to the Okayama-Berg method (Okayama H. and P. Berg, Mol. Cell Biol. , 2 , 161, 1982; and Mol. Cell Biol. , 3 , 280, 1983). As a vector, an oligo dT-tailed (T = 17) plasmid primer (pcDV-1) and an oligo dG-tailed (G = 12) pL-1 Hind III linker were used. More specifically, 8µg of the above-mentioned poly A (+) RNA, 2 µg of the dT-tailed plasmid primer (pcDV-1), 2 mM of four dNTPs including ³⁵S-dCTP, and 20.6 units of a reverse transcriptase (RTase) were incubated in 40 µl of a reaction mixture containing 100 mM of Tris-HCl (pH 8.3), 10 mM of MgCl₂ , 140 mM of KCl, 2 mM of methylmercury hydroxide, 20 mM of mercaptoethanol, and 1 mM of RNase inhibitor at 42°C for 60 minutes, to form a cDNA-plasmid. To the reaction mixture, 3.5 mM of dCTP and 30 units of terminal transferase were added, resulting in the formation of 73.5 µl total volume of the reaction mixture. The reaction mixture was subjected to reaction at 37°C for 3 minutes, and 2.5 mM of EDTA containing 0.5% (final concentration) of SDS was added to the reaction mixture to terminate the reaction. The reaction mixture was extracted with chloroform and phenol, and the DNA was then precipitated with ethanol. The thus-prepared cDNA-plasmid was digested with 20 units of Hind III in 40 µl of a buffer containing 10 mM of Tris-HCl (pH 7.5), 50 mM of NaCl, 10 mM of MgCl₂ and 1 mM of dithiothreitol, at 37°C for 2 hours. Then, 120 mg of the digested cDNA-plasmid and 35 µg of the PL-1 linker were incubated in 40 µl of a reaction mixture containining 0.1 M NaCl, 10 mM Tris-HCl (pH 7.8) and 1 mM EDTA, first at 65°C for 5 minutes and that of 42°C for 60 minutes, and the reaction mixture was then cooled to a room temperature to allow annealing. After the reaction mixture was cooled to 0°C, 0.3 µg of E. coli DNA ligase was then added to the reaction mixture, and the reaction mixture was incubated at 12°C overnight. Then, 200 mg of E. coli DNA ligase, 0.450 units of RNase H and 0.8 units of DNA polymerase were added to the reaction mixture to make the total volume of the mixture 48 µl, and the temperature of the reaction mixture was then increased stepwise to 12°C, 15°, 20°C, and to 25°C, for 30 minutes at each temperature, resulting in digestion of an mRNA region, formation of a double-stranded cDNA and ligation of the resulting cDNA, to complete the construction of plasmids containing the cDNA. The thus-obtained plasmids were used to transform Escherichia coli DH-5 and E. coli MC1061, and a cDNA library comprising 7,700 clones and a cDNA library comprising 7,300 clones (total 15,000 clones) were obtained from E. coli DN-5 and E. coli MC1061, respectively. (5) Screening of cDNA Library From among the above-mentioned 15,000 clones, 109 clones were screened by the colony hybridization method. As a result, a clone hybridizing with the SSPE-H-cDNA probe and a clone hybridizing with CDV-F-cDNA were obtained. These clones were designated as Escherichia coli DH-RVH and Escherichia coli DH-RVF respectively; and plasmids in these E. coli cells were designated as pDH-RVH and pDH-RVH. The size of cDNA insert (designated as RV-H) in the plasmid pDH-RVH was about 2.2 kb, and the size of the cDNA insert (designated as RV-F) in the plasmid pDH-RVF was about 1.6 kb. It was considered that these cDNA inserts contained approximately the entire coding regions for the H protein and F protein of the rinderpest virus, respectively. The above-mentioned Escherichia coli DH-RVH was deposited with the Fermentation Research Institute Agency of Industrial Science and Technology (FRI), Higashi 1-1-3, Yatabe-cho, Tsukuba-gun, Ibaraki-ken, Japan, as FERM BP-1165 under the Budapest treaty on August 29, 1986. The above-mentioned Escherichia coli DH-RVF was deposited with the FRI as FERM BP-1164 under the Budapest treaty on August 29, 1986. By repeating the same procedure as described above, a clone hybridizing with the SSPE-H-cDNA probe and a clone hybridizing with the CDV-F-cDNA were obtained. These clones were designated as Escherichia coli DH-RVH-2 and Escherichia coli DH-RVF-2, respectively; and plasmids in these E. coli cells were designated as pDH-RVH-2 and pDH-RVF-2, respectively. The size of the cDNA insert (designated as RV-H-2) in the plasmid pDH-RVH-2 was about 3.2 kb, and the size of the cDNA insert (designated as RV-F-2) in the plasmid pDH-RVF-2 was about 2.4 kb. The RV-H-2 contained an entire coding region for the H protein of the rinderpest virus, and the RV-F-2 is considered to contain an entire coding region for the F protein of the rinderpest virus. The above-mentioned Escherichia coli DH-RVH-2 was deposited with the FRI as FERM BP-1319 under the Budapest treaty on March 24, 1987. The above-mentioned Escherichia coli DH-RVF-2 was deposited with the FRI as FERM BP-1318 under the Budapest treaty on March 24, 1987. (6) Restriction enzyme cleavage map The restriction enzyme cleavage sites of the above-mentioned cDNA inserts were analyzed to determine restriction enzyme cleavage maps, according to a conventional procedure. Figure 1 represents the restriction enzyme cleavage maps for the cDNA inserts RV-H and RV-F, and Fig. 2 represents the restriction enzyme cleavage maps for the cDNA inserts RV-H-2 and RV-F-2. (7) Determination of nucleotide sequences of cDNAs coding for H protein and F protein The plasmids pDH-RVH and pDH-RVH-2 were digested with restriction enzymes Bam HI, Pvu II, Pst I, Eco RI, Hind III, and Sma I, and DNA fragments of several hundred base pairs in length were separated. Each DNA fragment was inserted in to a polylinker site of a phage M13 DNA, and the nucleotide sequence was determined according to the Sanger dideoxy chain termination method. The result is shown in Figs. 3-1 to 3-4. According to a similar procedure, the nucleotide sequence coding for F protein was determined. The result is shown in Figs. 4-1 to 4-5. (8) Expression of gene COS7 cells were cultured in a cell culture chamber in 1 ml of Eagl's minimum essential medium containing 10% felal calf serum at 37°C for 24 hours. The DNA of the plasmid pDH-RVH-2 was coprecipitated with calcium phosphate, and 1 µg of the precipitated DNA was transfected in to the cultured COS7 cells. The cells were then treated with a DMEM medium containing 10% dimethylsulfoxide and 5% fetal calf serum. fresh medium was added to the cells, and culturing was carried out at 37°C for two to three days. The cells were then fixed with acetone, and were observed by an indirect fluorescent antibody technique using an antibody specific to the rubeola virus. In this technique, an antigen H reactive with the antibody was observed on the cells. In same manner, it was confirmed that the pDH-RVF and pDH-RVF2 expressed an antigen F. The present cDNA coding for the hemagglutinin protein of the rinderpest virus, and the cDNA coding for the fusion protein of the rinderpest virus are useful as gene starting materials in a process for the construction of a recombinant virus by inserting them into, for example, a vaccinia virus, or in a process for the production of antigen proteins by expressing the cDNA in E. coli , yeast or animal cells. Moreover, the present cDNA and fragments thereof are useful as hybridization probes for screening genes of different origin but containing a similar nucleotide sequence, such as cDNA of different origin. Moreover the present cDNA and fragments thereof are useful as components of a kit for the detection of the rinderpest virus and related viruses. It should be noted that the procedures disclosed in the present specification can be applied to the cloning of other cDNAs coding for an antigen protein or a part thereof of the rinderpest virus. Such cDNA's are also covered by the present invention. It will also be appreciated that the ambit of the invention is not limited to the precise DNA sequences disclosed herein. For obvious reasons of, inter alia, codon degeneracy and the uncertainty of paratopic/epitopic site definition, the invention also includes, in addition to the precise sequences, DNA sequences hybridizable with the aforesaid sequences and encoding part at least of a rinderpest virus antigenic polypetide or DNA sequences as aforesaid modified by the deletion or addition of one or more codons or the modification of one or more codons but still encoding a rinderpest virus antigenic polypeptide.

Claims: DNA coding for the entirety or part of an antigen protein of a rinderpest virus wherein said antigen protein is: a) A hemagglutinin protein encoded by the nucleotide sequence: or a DNA sequence hybridizable with the aforesaid sequence and encoding part at least of a rinderpest virus antigenic polypeptide or a DNA sequence as aforesaid modified by the deletion or addition of one or more codons or the modification of one or more codons but still encoding a rinderpest virus antigenic polypeptide or b) a fusion protein encoded by the nucleotide sequence: or a DNA sequence hybridizable with the aforesaid sequence and encoding part at least of a rinderpest virus antigenic polypeptide or a DNA sequence as aforesaid modified by the deletion or addition of one or more codons or the modification of one or more codons but still encoding a rinderpest virus antigenic polypeptide. The use of DNA according to claim 1 in the preparation of a vaccine for a rinderpest virus. The use of DNA according to claim 1 (a) in constructing a recombinant virus, (b) in producing antigen protein, (c) as a hybridization probe, or (d) in a kit for the diagnosis or detection of rinderpest virus or related viruses. A plasmid or other expression vector incorporating DNA according to claim 1 or a host cell incorporating said plasmid or vector in its expressible genone. A plasmid or vector or cell according to claim 4 and obtainable from FERM BP-1164, FERM BP-1165, FERM BP-1318, or FERM BP-1319.

Inventors: SUGIMOTO, MASANOBU, YAMANOUCHI, KAZUYA, YOSHIKAWA, YASUHIRO
Applicants: TONEN CORPORATION
Application year: 1987
Main classification: C12N15
IPC codes: A61K39, C12R1, C07K14, C12N15, C12Q1, C07K16