WO2015149386A1 - 狂犬病病毒ctn鸡胚细胞适应株 - Google Patents

狂犬病病毒ctn鸡胚细胞适应株 Download PDF

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WO2015149386A1
WO2015149386A1 PCT/CN2014/075230 CN2014075230W WO2015149386A1 WO 2015149386 A1 WO2015149386 A1 WO 2015149386A1 CN 2014075230 W CN2014075230 W CN 2014075230W WO 2015149386 A1 WO2015149386 A1 WO 2015149386A1
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virus
vaccine
rabies
ctn
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王春华
郭采平
罗姗
刘永娣
李慧
丁玉江
容伟华
周兰贞
周维
宋清爽
黄伟荣
田华
朱士茂
张信
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深圳市卫光生物制品股份有限公司
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Definitions

  • the invention relates to a novel rabies virus strain, in particular to a rabies virus chicken embryo cell adaptation strain.
  • Rabies virus Virus is a highly neurotropic virus, Rhabdoviridae rabies virus (Lyssavirus) Genius) A single-stranded negative-strand RNA virus that does not divide into segments, can cause zoonotic global infectious disease - rabies. It is reported that there are about 55,000 deaths due to rabies worldwide each year, and the actual number of deaths should be significantly higher than the statistics (http://www.worldrabiesday.org/). At present, except for rabies in a few countries and regions such as Japan, the United Kingdom, and Hawaii, the disease is worldwide, and Asia and Africa are the most severe areas of human rabies, accounting for 99% of the total number of deaths worldwide.
  • RV genome size is about 12 Kb, from the 3' end to the 5' end, five RV structural protein genes are arranged in sequence, encoding five known structural proteins: nuclear protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L).
  • N nuclear protein
  • P phosphoprotein
  • M matrix protein
  • G glycoprotein
  • L RNA-dependent RNA polymerase
  • N protein binds to RNA to form ribonucleic acid protein (RNP) during RV replication; L protein and phosphoprotein are closely linked to RNP in a helical structure, ensuring transcription and replication of the genome in the cytoplasm; M protein is a connexin RV One of the five structural proteins with a large variation, occupying the position between the nucleocapsid and the outer shell, and joining the two together; the G protein is the only exposure of the RV to the surface of the virion and induces host neutralization.
  • the protein of the antibody whose carboxy terminus is inserted into the RNP, is responsible for binding to the receptor on the surface of the host cell.
  • the G protein is encoded by the G gene and has a total of 1675 nucleotides encoding 524 amino acids.
  • G protein is the main protective antigen of RV, which can induce the body to produce neutralizing antibodies and protect the body against RV infection (Wiktor Et al., 1973. J. Immunol. 110: 269-276; Cox et Al., 1977. Infect. Immun. 16: 754-759; Perrin et Al., 1985. Vaccine. 3: 325-332).
  • RV the main protective antigen of RV
  • G protein There are at least 3 neutralizing antibody binding sites on the G protein: antigenic site III (antigen) III, GIII), antigenic site II (antigen II, GII) and secondary antigenic site I (antigen I, GI), wherein GII is located in the 34-200 amino acid segment and GIII is located in the amino acid segment 330-357 (Tordo N., 1996. Laboratory techniques In rabies.4th ed.WHO: 28-49). GII and GIII not only induce the production of epitopes of neutralizing antibodies, but are also an essential part of G protein folding and transport.
  • G protein is mainly caused by the substitution of amino acids 34-42, 147, 184, 198-200 at the GII site and 330-340 amino acids at the GIII site (Benmansour). Et al., 1991. J. virol. 65: 4198-4203).
  • a single amino acid change on the G protein can alter the antigenicity of the RV. Irie et al.
  • RV The replacement of aspartic acid and methionine by the lysine at position 330 and the arginine at position 333 in the extracellular region of G glycoprotein significantly reduce the interaction between glycoprotein and neuroblastoma cells, and form a double The mutant RV strain is not pathogenic to adult mice.
  • Other studies have also found that when the 333th arginine of the G protein is replaced by isoleucine, glutamine, glutamic acid or glycine, the mutant strain is infected regardless of the dose and route. No pathogenicity (Dietzschold B. et al., 1983. Proc. Natl. Acad. Sci. U.S.A., 80: 70-74; Seif et al., 1985. J.
  • RV is a neurotropic virus, it is invasive to almost all mammalian nervous tissues.
  • the world's first rabies vaccine was developed using RV-infected rabbit spinal cord, and then cultured in rabbit brain, mouse brain, sheep brain and other animal tissues to prepare a vaccine.
  • WHO World Health Organization
  • PCEC chicken embryo cell vaccine
  • PVRV purified vero cell rabies vaccine
  • RV The CTN-1 strain is a strain approved by the WHO and relevant Chinese authorities for the production of rabies vaccine. It was built and preserved by the China Food and Drug Administration. In the 1980s, Li Hongling and others will RV CTN-1 strain was cultured in vero cells for adaptive passage, resulting in higher titer RV Vero cell-adapted strains can be used for vaccine production (Li Hongling et al. 1989. Journal of Biological Products. 2: 22-25). Dong Guanmu et al further confirmed that CTN-1 strain can be quickly adapted in vero cells, and the titer can reach 7.0. LogLD50/ml or more, and the virus culture solution can be harvested multiple times (Dong Guanmu et al. 1995. Progress in Microbiology and Immunology.
  • Rudolph Barth et al. (US 4115195) describes the process of producing a rabies vaccine in which a variety of RV strains can utilize chicken embryo fibroblasts (Chicken embryo fibroblast) Cells, CEC) Preparation of rabies vaccines, such as VP11 strain, Pasteur strain, PM strain, Flury LEP and Flury HEP. They specifically provide examples of infection of CEC using RV immobilized strains VP11, Flury LEP, and Flury HEP.
  • CEC chicken embryo fibroblasts
  • PATEL Pradip Maganlal and PATEL Pankaj Ramanbhai (PCT/IN2008/000262) describe Pitman The moore strain (Wistar strain PM-HDCS ⁇ 1503-3M) is adapted to CEC, and the obtained virus strain has high yield and short production time, and is easy to be produced on a large scale.
  • the invention provides a rabies virus CTN chicken embryo cell adapting strain which can rapidly proliferate on CEC, has high virus titer and good immunogenicity, but has greatly reduced virulence to adult mice; using rabies virus CTN chicken embryo Cell-adapted strains produce rabies vaccine, which not only has good immunogenicity and protective effect, but also is more reliable in terms of safety. It is an ideal strain for producing rabies vaccine; in addition, rabies virus CTN chicken embryo cell-adapted strain has good immunoprotective properties. The immunogen stimulates the body to produce higher levels of antibody levels for the preparation of high quality anti-RV antibodies.
  • the invention provides a rabies virus CTN chicken embryo cell adaptation strain, and the microorganism preservation number is: CGMCC No. 6510;
  • the present invention also provides the use of the rabies virus CTN chicken embryo cell adaptation strain in the preparation of a rabies virus inactivated vaccine.
  • the present invention has the following features:
  • the present invention provides a novel RV strain designated as RV CTN chicken embryo cell adaptation strain, ie RV CTNCEC25 strain.
  • the strain has a new base and amino acid sequence, can produce high virus titer on CEC, has high immunogenicity, can be used for producing rabies vaccine, preparing anti-RV antibody and detecting RV antibody titer.
  • the invention determines five structural proteins and whole gene sequences of the strain.
  • the present inventors used molecular biology methods to analyze five structural protein genes and whole gene sequences of the strain, and found that the five structural proteins of the strain have different degrees compared with the female CTN-1 strain. Variation, in which the G protein is the most variable.
  • nucleotides of the G protein coding sequence 6 are located in the coding sequence region of the G protein (Coding Sequences, CDS), they are 3812 (A ⁇ G), 4371 (G ⁇ A), 4538 (G ⁇ A), 4635 (C ⁇ A), 4636 (A ⁇ G) and 4826 (T ⁇ C), the corresponding amino acids are 147 (Lys ⁇ Glu), 333 (Arg ⁇ Gln), 389 (Glu-Lys), 421 (Pro ⁇ Gln) and 485 (Ser ⁇ Pro) .
  • Another 5251 nucleotide (C ⁇ A) is located in the G protein link. The sequence region does not affect the amino acid sequence of the G protein.
  • the invention provides an inactivated vaccine of the RV CTNCEC25 strain.
  • the virus solution can be harvested after more than 80% of the cytopathic changes.
  • the original vaccine was prepared by inoculating ⁇ -propiolactone according to the ratio of inactivating agent to viral solution 1:4000 (v/v), and used for immunizing mice of 12-14 g for immunogenicity test and efficacy test. .
  • the results showed that the strain had good immunoprotective properties, and its efficacy could reach the current marketed vaccine level and met the pharmacopoeia standards.
  • Figure 1 shows the passage history of the RV CTNCEC25 strain.
  • Figure 2 shows the cell morphology of RV CTNCEC25 strain after inoculation of primary chicken embryo fibroblasts.
  • Figure 3 shows the specific fluorescence of the RV CTNCEC25 strain.
  • Figure 4 shows the primers used for the whole gene sequencing of the RV CTNCEC25 strain.
  • the virus samples to be tested were serially diluted 10 times with PBS, and then serially diluted 3 times. Then, 50 ⁇ l of the diluted virus solution was used to inoculate 50 ⁇ l of BSR cells having a cell density of 1 ⁇ 10 6 cells/ml (from the Institute for Viral Disease Control and Prevention of the Chinese Center for Disease Control and Prevention). After mixing well, it was placed at 37 ° C, 5% CO 2 for 24 hours.
  • the V5 strain is a mother strain. Through a comparison of several passage pathways and techniques, it was found that the strains obtained by passage of the following routes can gradually adapt to the growth in chicken embryo cells.
  • the rabies virus chicken embryo cell adaptation strain was named as CTNCEC25 strain.
  • Step 1 The CTN-1V5 strain was passaged on vero cells to increase the virus titer.
  • RV CTN-1V5 (derived from the China Food and Drug Control Institute, the 5th generation of CTN-1 strain vero cells) was diluted 10 times with PBS (pH 7.4), and then according to 1:100 ⁇ 1:1000 Proportional inoculation of vero monolayer cells (from the China Food and Drug Control Institute, 121 generations), supplemented with cell maintenance solution after 60 minutes of adsorption at 37 °C (based on 199 medium, adding a final concentration of 10% (v/v) Bovine serum, pH 7.2 ⁇ 8.0), placed in a 37 ° C, 5% CO 2 incubator for static culture, cultured for 4 to 6 days to harvest the virus supernatant, so continuous transmission for 10 generations, the virus titer of up to 7.5 CTN-1V15 strain with lgLD 50 /ml or more.
  • Step 2 The CTV-1V15 strain was passed through the chicken embryo for one generation, and the RV CTN chicken embryo generation virus was obtained.
  • the obtained CTN-1V15 strain was diluted with PBS (pH 7.4) 1:10 to 1:1000, and the virus dilution was inoculated into the SPF chicken embryo of 6-7 days old by the yolk sac, 0.5 ml per embryo.
  • Step 3 The RV CTN chicken embryo generation virus was passaged in chicken embryo fibroblasts (CEC) to gradually adapt to CEC.
  • CEC chicken embryo fibroblasts
  • the RV CTN chicken embryo generation virus (CTNCE01) obtained in the second step was appropriately diluted with PBS (pH 7.4) (10 0 to 10 -4 ), according to different inoculation ratios (1:10 to 1:5 ⁇ 10 5 ). Mix well with the prepared CEC suspension (M0, cell density 0.8-1.4 ⁇ 10 6 cells/ml), place in a cell culture flask, and place in a 35-37 ° C, 5% CO 2 incubator. to cultivate. The virus was cultured until the cells showed lesions (mainly characterized by clustering of diseased cells, rounding, faster aging and shedding), and the virus was further passaged on CEC according to the above culture conditions.
  • the virus titer was sampled for each generation of virus using the Fluorescence Focus Units Assay (FFU, specific operation as follows). After 30 to 45 infection and adaptation tests, it was obtained in CEC. Satisfactory virus titer results, the five structural protein genes were consistent by sequencing, and it was confirmed that the strain was an RV chicken embryo cell-adapted strain. The strain was named RV CTNCEC25 strain, and the 33-generation strain was collected in China. General Microbiology Center of the Microbial Culture Collection Management Committee. The passage history of the poisonous strain of the present invention is shown in Fig. 1.
  • the other culture conditions were the same as above, and the serial batch was passed for 3 generations to obtain the main seed batch. Toxic species.
  • the main seed batch was continued to be transplanted for 4 generations in CEC under the culture conditions for preparing the main seed batch, and the working seed batch was obtained.
  • the original seed batch virus obtained by the invention has a titer of not less than 6.5 lgFFU/ml, the titer of the main seed batch and the working seed batch obtained from the original seed is not less than 7.0 lg FFU/ml.
  • the relevant deposit information of the RV CTNCEC25 strain of the present invention is as follows:
  • CGMCC The depositary institution
  • Taxonomy A novel strain belonging to the family Rhabdoviridae and Lyssaviruses.
  • the above RV CTNCEC25 strain has the following biological characteristics:
  • the cells were cultured in a 36 ° C, 5% CO 2 incubator. After more than 80% of the cells showed lesions, 0.5 ml of the virus supernatant was aspirated by a disposable pipette, and the titer was determined by the FFU method. As a result, it was found that the infectious titer of the virus was 7.0 to 8.0 lg FFU/ml 72 to 120 hours after the virus was inoculated.
  • the SPF-class Kunming suckling mice of 1 to 3 days old were challenged with the viral amount of 100 LD 50 of the RV CTNCEC25 strain in the brain, and the parental strain CTN-1 strain was treated as the control, and observed for 14 days. As a result, it was found that more than 90% of the suckling mice that were attacked by the strain of the strain died, and 100% of the suckling mice attacked by the parental CTN-1 strain died. It indicated that RV CTNCEC25 strain still has strong intracerebral virulence and lethality in suckling rats.
  • the RV CTNCEC25 strain and the RV CTN-1 strain virus solution with the highest dose of 10 6.5 LD 50 were injected into the SPF Kunming with different specifications (11-13 g, 18-20 g and 28-30 g) by subcutaneous, intraperitoneal and intramuscular routes, respectively.
  • Albino mice 10 mice of different routes and different specifications, were observed for 21 days.
  • RV CTNCEC25 strain was injected into the mouse subcutaneously, intraperitoneally or intramuscularly, and the mice did not develop disease.
  • RV CTN-1 the mice in the injection group had rabies symptoms and death.
  • Table 2 It was shown that the RV CTNCEC25 strain completely lost its pathogenicity to the subcutaneous, muscle and abdominal cavity of adult mice compared with its parental strain RV CTN-1.
  • the neutralization test was performed by the FFU method to identify the specificity of the RV CTNCEC25 strain.
  • the specific method is as follows:
  • the RV CTNCEC25 strain was first diluted 10 times with PBS, then diluted 3 times, and then the appropriate dilution of the virus dilution (general test group took 3 -1 ⁇ 10 -1 ⁇ 3 -4 ⁇ 10 -1 dilution, the control group took 3 -1 ⁇ 10 -4 ⁇ 3 -4 ⁇ 10 -3 dilution) and the dilution of 10 -2 rabies virus-specific immune serum (from Wuhan Institute of Biological Products, The titer of 200 IU/ml) (test group) and the dilution of 10 -2 negative horse serum (derived from Guangzhou Ruite Biotechnology Co., Ltd.) (control group) were mixed in equal amounts and placed in a 37 ° C water bath for 60 minutes.
  • Neutralization index control titer (FFU / ml) / test group titer (FFU / ml)
  • the neutralization index of the strain reached more than 1000, which was in accordance with the provisions of the 2010 edition of the Chinese Pharmacopoeia, "the neutralization index is not less than 500", indicating that the strain is an RV strain.
  • ⁇ -propiolactone was slowly added so that the final ratio of ⁇ -propiolactone to the crude virus solution reached 1:4000 (v /v), stir well and mix at 2-8 °C for incubation. It is necessary to incubate for at least 24 hours to fully inactivate virus infectivity without losing the antigenicity of the virus. After 24 hours, the ⁇ -propiolactone-inactivated virus solution was placed in a water bath at 37 ° C for 2 hours to sufficiently hydrolyze the residual ⁇ -propiolactone in the virus solution to obtain a test original vaccine.
  • the original vaccine obtained in the above step was examined for immunoprotection according to the 2010 edition of the Chinese Pharmacopoeia.
  • the specific experimental results are shown in Table 4.
  • the test showed that the protection index of the strain can reach more than 50,000, which is far greater than the provisions of the 2010 edition of the Chinese Pharmacopoeia, "the neutralization index is not less than 500", indicating that the virus has good immunoprotective properties.
  • RV was designed based on the sequence of the RV CTN-1 strain published by the NCBI website Genbank.
  • the whole genome-specific sequencing primers of CTNCEC25 strain were synthesized by Invitrogen Biotechnology Co., Ltd., as shown in Figure 4.
  • the whole genome sequence of the strain was amplified by RT-PCR, sequenced and spliced (completed by Invitrogen Biotechnology Co., Ltd.), and the genome sequence was analyzed using the MegAlign tool in DNAStar software.
  • the nucleotide sequence of the strain obtained the amino acid sequence of five known structural proteins of the strain of the present invention.
  • the full length of the strain was 11924 bp (Genbank Accession: KJ466147), wherein the N gene contains 1425 nucleotides, consisting of 450 amino acids; the P gene contains 989 nucleotides, consisting of 297 amino acids; the M gene contains 803 nucleotides, consisting of 202 amino acids; G The gene contains 2067 nucleotides and consists of 524 amino acids; the L protein contains 6474 nucleotides and consists of 2128 amino acids.
  • the full-gene sequence of the RV CTNCEC25 strain is as follows:
  • amino acid sequences of the five structural proteins of the RV CTNCEC25 strain are as follows:
  • RV CTNCEC25 strain working seed batch virus ie CTNCEC25 strain 40 generation virus species
  • CEC suspension was prepared as described in Example 1, and the medium was changed to M2 or M3, whose cell number was adjusted to 0.8 to 1.4 ⁇ 10 6 cells/ml, was uniformly mixed, and then dispensed in a cell bottle or a cell factory, and then cultured at 33 to 36 ° C, 5% CO 2 .
  • the virus supernatant can be harvested after more than 80% of the cells have lesions, and the harvested virus supernatant is filtered and clarified by a pinhole filter ( ⁇ 0.60 ⁇ m, Millipore), and ⁇ -propiolactone is added to make it
  • the final ratio of the virus suspension is 1:4000 (v/v), fully mixed and mixed, placed in 2-8 Incubate at °C for at least 24 hours to ensure sufficient inactivation of the virus's infectivity without loss of antigenicity of the virus. After 24 hours, the fully inactivated virus solution was placed at 37 ° C for 2 hours to fully hydrolyze the residual ⁇ -propiolactone in the virus solution to obtain the vaccine to be tested, and stored in 2-8. °C, for vaccine titer and serum antibody levels.
  • the NIH method is used to test the titer of the vaccine to assess its immunoprotective properties.
  • the prepared vaccine to be tested was also subjected to 5-fold gradient dilution with PBS, and finally the mice were immunized with a dilution ranging from 1:25 to 1:3125.
  • mice Take the diluted reference vaccine and the vaccine to be tested, and immunize the mice by the intraperitoneal route, at least 16 mice per dilution, 0.5 ml vaccine dilution per mouse. Seven days after the first immunization, the mice were subjected to secondary immunization according to the steps and amounts of the primary immunization. 14 days after the initial immunization, immunization with a suspension of CVS virus containing 5 to 100 LD 50 (from the China Food and Drug Administration, initially CVS-8, passed through the mouse brain for 2 generations, ie CVS-10) The latter mice were challenged intracerebally. After intracerebral challenge, 14 days were observed day by day, and the dead mice within 3 days were recorded as non-specific death. The titer is calculated by the Reed-Muench method. Find:
  • the vaccine reference and test vaccine actual ED 50 values are given in the experimental animals between the highest and lowest dose.
  • the attack amount of the CVS strain virus suspension was 10 LD 50 /0.03 ml, and all the experimental animals receiving the attack virus suspension showed death.
  • the titer of the test vaccine is calculated according to the following formula:
  • T is the reciprocal of the ED 50 of the vaccine to be tested
  • S is the reciprocal of the reference vaccine ED 50 ;
  • d T is the first human dose of the vaccine to be tested, ml;
  • d S is the first human dose of the reference vaccine, ml;
  • D is the titer of the reference vaccine, IU/ml.
  • the average titer of the vaccine to be tested is 5.39 IU/ml, and the challenge dose of the CVS strain applied in each test is between 5 and 100 LD 50 . Every reference vaccine applied to detect the actual ED 50 are located between the highest and lowest dilution. Therefore, it can be concluded that the vaccine is immunoprotective and the NIH titer is higher than the quality standard of 2.5 IU/dose of WHO and the current Chinese Pharmacopoeia.
  • the vaccine to be tested is prepared as an immunogen, and the abdominal cavity is used for 12 to 14
  • the SPF-class albino Kunming mice were initially immunized with 0.5 ml per mouse, and 7 days after the first immunization, the mice were subjected to secondary immunization according to the steps and amount of the primary immunization, 3 days and 7 days after the second immunization. Five mice were taken for 10 days and 14 days, blood samples were collected through the arterial artery, and the collected blood samples were incubated at 37 ° C for 1 hour to coagulate, and then the blood samples after coagulation were 2000.
  • the serum was separated by centrifugation at rpm for 10 minutes, and the serum was aseptically collected in a sterile test tube or a cryotube.
  • the collected serum was inactivated at 56 ° C for 30 minutes and stored at -70 ° C until use.
  • the antibody titer was determined by the above collected serum, and the antibody titer was determined according to the 2010 edition of the Chinese Pharmacopoeia Appendix XI.
  • the "rapid fluorescent focus inhibition test method" in J "rabies immunoglobulin titer assay” was carried out. The measurement results are shown in Table 6:
  • the original vaccine prepared by the strain of the present invention is used as an immunogen to immunize mice, and the average antibody titer of the serum in the mice of 10 days and 14 days after the second immunization can reach 40. Above IU/ml.
  • the vaccine to be tested is prepared as an immunogen, and the muscle route is 1.5 to 2.0.
  • Five rabbits of kg were initially immunized, 1.0 ml per rabbit, 7 days after priming, secondary immunization of rabbits according to the steps and amount of primary immunization, 14 days after secondary immunization through ear veins
  • Blood samples were collected, and the collected blood samples were incubated at 37 ° C for 1 hour to coagulate, and then the blood samples after coagulation were 2000.
  • the serum was separated by centrifugation at rpm for 10 minutes, and the serum was aseptically collected in a sterile test tube or a cryotube.
  • the collected serum was inactivated at 56 ° C for 30 minutes and stored at -70 ° C until use.
  • the antibody titer was determined by the above collected serum, and the antibody titer was determined according to the 2010 edition of the Chinese Pharmacopoeia Appendix XI.
  • the "rapid fluorescent focus inhibition test method" in J "rabies immunoglobulin titer assay” was carried out. The results are shown in Table 7:
  • the original vaccine prepared by the strain of the present invention is used as an immunogen to immunize rabbits, and the average antibody titer of the serum in the rabbits after 14 days of secondary immunization can also reach 40. Above IU/ml.
  • the antigen prepared by the strain of the present application is used as an immunogen, a higher level of anti-RV serum can be obtained from 10 days to 14 days after the second immunization of the animal, and preparation of the antiserum or antibody can be performed.

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Abstract

提供了一种保藏号为：CGMCCNo.6510的狂犬病病毒CTN鸡胚细胞适应株，及其在制备狂犬病病毒灭活疫苗中的用途。

Description

狂犬病病毒CTN鸡胚细胞适应株 狂犬病病毒CTN鸡胚细胞适应株
技术领域
本发明主要涉及一种新的狂犬病病毒株，具体涉及一种狂犬病病毒鸡胚细胞适应株。
背景技术
狂犬病病毒（Rabies virus，RV）是高度嗜神经病毒，为弹状病毒科（Rhabdoviridae）狂犬病病毒属（Lyssavirus genus）中不分节段的单股负链RNA病毒，能引起人兽共患的世界性传染病—狂犬病。据报道全世界每年因狂犬病的死亡人数约有5.5万例,实际死亡人数应明显高于该统计数字（http://www.worldrabiesday.org/）。目前除日本、英国、夏威夷等少数国家和地区没有狂犬病发生以外,该病呈世界性流行，其中亚洲和非洲是人狂犬病发生最严重的地区,占全世界死亡总人数的99%。在正式报告狂犬病的国家中,印度每年有3万人以上死于狂犬病,居首位；我国近20年每年统计到的死亡人数超过3000例,居第二位(Yunpeng wang et al.,2012.Journal of Applied Virology.1:10-19)。目前狗仍然是人狂犬病的最重要的宿主。
RV基因组大小约12 kb，由3′端至5′端依次排列着5个RV结构蛋白基因，分别编码五种已知的结构蛋白：核蛋白（N）、磷蛋白（P）、基质蛋白（M）、糖蛋白（G）和依赖RNA的RNA多聚酶（L）。N蛋白在RV复制过程中与RNA结合成核糖核酸蛋白（RNP）；L蛋白和磷蛋白与RNP紧密相连呈螺旋状结构，确保基因组在细胞质中转录和复制；M蛋白是一种连接蛋白，为RV 5种结构蛋白中变异较大的蛋白质之一，其占据了核衣壳和外壳之间的位置，并将两者连接一起；G蛋白是RV中唯一暴露在病毒粒子表面和诱导宿主产生中和抗体的蛋白，其羧基端插入RNP中，负责与宿主细胞表面的受体的结合。
G蛋白由G基因编码，共有1675个核苷酸，编码524个氨基酸。G蛋白是RV的主要保护性抗原，能诱导机体产生中和抗体，保护机体抵抗RV的感染(Wiktor et al.，1973.J.Immunol.110:269-276；Cox et al.，1977.Infect.Immun.16:754-759；Perrin et al.，1985.Vaccine.3:325-332)。当前无论人用还是兽用狂犬病疫苗,其效价主要取决于疫苗制剂中G蛋白的含量。G蛋白上至少存在3个中和抗体结合位点：抗原位点Ⅲ（antigen Ⅲ，GⅢ）、抗原位点Ⅱ（antigen Ⅱ，GⅡ）及次要抗原位点Ⅰ（antigen Ⅰ，GⅠ），其中GⅡ位于34-200氨基酸区段，GⅢ位于330-357位氨基酸区段（Tordo N.,1996.Laboratory techniques in rabies.4th ed.WHO：28-49）。GⅡ和GⅢ不但是诱导产生中和抗体的抗原表位，而且也是G蛋白折叠和运输的必需部分。研究表明G蛋白抗原性变化主要是由GⅡ位点上34-42位、147位、184位、198-200位氨基酸以及GⅢ位点上330-340位氨基酸的替换引起（Benmansour et al.，1991.J. virol. 65:4198-4203）。G蛋白上单一氨基酸的改变就能改变RV的抗原性。Irie等用单克隆抗体研究RV G蛋白上的1个构象表位时发现，第36位苏氨酸被脯氨酸替换形成的突变株丢失了这一抗原表位，单克隆抗体不能中和该突变株；第39位丝氨酸被苏氨酸替换形成的突变株虽能与单克隆抗体结合，但其空间构象发生了改变，单克隆抗体也不能中和突变株（Irie T et al.,2002.Microbiol. Immunol.46:449-461）。其他研究者利用抗GⅡ抗原位点的单克隆抗体研究G蛋白的抗原性也得到过类似的结果。G蛋白的结构与RV毒力有关。研究发现，当RV G糖蛋白膜外区330位的赖氨酸和333位的精氨酸分别被天门冬氨酸和甲硫氨酸取代会明显降低糖蛋白和神经瘤细胞之间的相互作用，且形成的双突变RV株对成年小鼠没有致病性。其他研究也发现，当G蛋白的第333位精氨酸被异亮氨酸、谷氨酰胺、谷氨酸或甘氨酸替换，不管以何种剂量、何种途径感染小鼠，该突变毒株均无致病性（Dietzschold B.et al.,1983.Proc. Natl. Acad. Sci. U.S.A.,80: 70-74；Seif et al., 1985. J.Virol. 53: 926-934；Tuffereau et al., 1989.Virology, 172: 206-212；Ito H. et al., 1994.Microbiol. Immunol. 38: 479-482: Coulon P et al., 1998.J. Virol. 72:272-274； Mutsuyo Takayama-Ito M. et al., 2006.Virus research, 119: 208-215）。此外G蛋白的第164-303位氨基酸与RV对成年鼠的致死性作用有关，其中第242、255、268位氨基酸能增强RV的毒力。最新的研究发现，无致病性毒株的第194位天冬酰胺被赖氨酸替换，能增强RV突变株的传播，提高RV突变株的毒力（Takayama-Ito M. et al., 2004.J.Neurovirol.10: 131-135）。
由于RV是嗜神经性病毒，几乎对所有哺乳动物的神经组织都具有侵染性。世界上首次试用的狂犬病疫苗就是用RV感染兔脊髓研制的，以后经兔脑、小鼠脑、羊脑等动物神经组织培养，制备疫苗。但由于该类疫苗存在严重的变态反应且效力低而被WHO停用（林放涛等.1992.狂犬病学.203-221；Meslin F.X. et al., 1996.Laboratory techniques in rabies.4th ed.WHO.223-313）。1956年Vienchange及其同事首次将RV在原代小鼠肾细胞上培养成功。2年后Kissling等将RV街毒株和固定毒株在原代地鼠肾细胞中传代（Kissing P.E.et al.,1958.Proc.Soc Exp Biol Med.98:223-225）。20世纪60年代以后用细胞制备疫苗有了很大发展，人们开始利用各种细胞来大规模生产狂犬病疫苗，特别是人二倍体细胞培养疫苗（HDCV）的研制（Wiktor et al.,1964.J Immunol.93:353-366）。由于不存在神经元组织，与之前的脑组织疫苗相比，组织培养疫苗不仅更安全，而且更有效。目前已批准若干种组织培养疫苗，其具有与HDCV相当的疗效和安全性。例如纯化鸡胚细胞疫苗（PCEC）（Barth etal.,1984.J Biol Stand.12:29-64;Schgal et al.,1993.J. Commun. Dis.27:36-43）和纯化vero细胞狂犬病疫苗（PVRV）(Suntharasamal et al.,1986.Lancet.2:129-131)。
RV CTN-1株是WHO和我国有关部门批准用于狂犬病疫苗生产的毒株，由中国食品药品检定研究院建株和保存。20世纪80年代李宏玲等将RV CTN-1株在vero细胞中培养进行适应传代，得到较高滴度的RV vero细胞适应株，可用于疫苗生产（李宏玲等.1989.生物制品学杂志.2:22-25）。董关木等进一步证实CTN-1株可在vero细胞内快速适应，滴度可到7.0 logLD50/ml以上，并可多次收获病毒培养液（董关木等.1995.微生物学免疫学进展.23:82-85）。目前已有多家生产单位应用RV CTN-1株生产vero细胞疫苗并取得生产文号和投产(俞永新.2008.狂犬病和狂犬病疫苗.第二版.中国医药科技出版社.207-209)。但vero细胞基质为永生性传代细胞系，因此有必要检测终产品中的细胞残留DNA（Ref. 欧洲药典.2004；中国药典.2010），因为其可能具有传递潜伏病毒和其他物质的风险。WHO也规定人用制品的残留DNA剂量应不超过10ng（WHO Expert Committee on Biological Standardition. Recommendations inactivated rabies vaccine for human use produced in cell substrates and embryonated eggs. Genava.WHO.2005）。
Rudolph Barth 等人（US 4115195）描述了生产狂犬病疫苗的过程，其中多种RV毒株均可利用鸡胚成纤维细胞（Chicken embryo fibroblast cells，CEC）制备狂犬病疫苗，如VP11株、Pasteur株、PM株、Flury LEP和Flury HEP。他们在专利中具体提供了利用RV固定株VP11、Flury LEP和Flury HEP感染CEC的实例。PATEL Pradip Maganlal和PATEL Pankaj Ramanbhai（PCT/IN2008/000262）描述了Pitman moore株（Wistar株PM-HDCS\1503-3M）适应于CEC，所得到的病毒株产量高且生产时间短，易于成规模生产。1984年底我国的陈道民和林放涛择用Flury（LEP）株68代鸡胚固定毒（兽用活疫苗株）适应到CEC培植人用狂犬病疫苗株，初步获得一株既能使用CEC又具有与a G株（3）免疫原性相仿的狂犬病鸡胚细胞适应株—武汉（Wuhan）34株（陈道民等.1988.中国人寿共患病杂志. 4:28-30）,但未见到有关该适应毒株进一步的报道。王远征等人也尝试将RV aG株在CEC上进行传代适应，得到滴度可达7.0 logLD50/ml的毒株，但该病毒株是否已完全适应于CEC尚不确定（王远征等.2012.中国生物制品学杂志.25:669-671）。迄今为止，还没有文献提及使CTN-1株适用于CEC。
发明内容
本发明提供了一种可在CEC上快速增殖，病毒滴度高、免疫原性好，但对成年小鼠的致病力大大减弱的狂犬病病毒CTN鸡胚细胞适应株；用狂犬病病毒CTN鸡胚细胞适应株生产狂犬病疫苗，不仅免疫原性和保护效果良好，而且在安全性方面更可靠，是生产狂犬病疫苗的理想毒株；此外狂犬病病毒CTN鸡胚细胞适应株具有良好的免疫保护性，可作为免疫原刺激机体产生较高水平的抗体水平，用以制备高质量的抗RV抗体。
本发明提供了一种狂犬病病毒CTN鸡胚细胞适应株，其微生物保藏号为：CGMCC No. 6510；本发明还提供了所述的狂犬病病毒CTN鸡胚细胞适应株在制备狂犬病病毒灭活疫苗中的用途。
总体来说，本发明具有以下特点：
第一方面，本发明提供了一种新的RV毒株，命名为RV CTN鸡胚细胞适应株即RV CTNCEC25株。该毒株具有新的碱基和氨基酸序列，能在CEC上产生高病毒滴度，具有较高的免疫原性，可用于生产狂犬病疫苗、制备抗RV抗体以及检测RV抗体效价等。
第二方面，本发明测定了该毒株的5个结构蛋白及全基因序列。本发明人利用分子生物学的方法，对该毒株的5个结构蛋白基因以及全基因序列进行分析，发现该毒株与母本CTN-1株相比，5个结构蛋白均有不同程度的变异，其中G蛋白变异最大。在G蛋白编码序列的7个变异的核苷酸中，6个位于G蛋白的编码序列区域（Coding sequences，CDS），它们分别是3812位（A→G）、4371位（G→A）、4538位（G→A）、4635位（C→A）、4636位（A→G）和4826位（T→C），对应的氨基酸分别是147位（Lys→Glu）、333位（Arg→Gln）、389位（Glu-Lys）、421位（Pro→Gln）和485位（Ser→Pro）。另外5251位核苷酸（C→A）位于G蛋白的link sequence区域，不影响G蛋白的氨基酸序列。
第三个方面，本发明提供了RV CTNCEC25株的灭活原疫苗。用本发明的RV CTNCEC25株毒种按照MOI（multiple of infection，MOI）=0.001～0.05 FFU/细胞的量接种CEC悬液，充分混悬后，置于33～35℃、5% CO2培养，待80%以上的细胞病变即可收获病毒液。然后按照灭活剂与病毒液1:4000(v/v)的比例加入β-丙内酯灭活制备原疫苗，用于免疫12～14 g的小鼠，分别进行免疫原性检查和效力测试。结果表明该毒株具有良好的免疫保护性，其效力可达到当前市售疫苗水平，符合药典标准。
附图说明
图1 显示RV CTNCEC25株的传代历史。
图2显示RV CTNCEC25株接种原代鸡胚成纤维细胞后的细胞形态。
图3显示RV CTNCEC25株的特异性荧光。
图4显示RV CTNCEC25株全基因测序所用的引物。
具体实施方式
以下多次涉及CEC的制备、细胞荧光灶转化单位实验（Fluorescence Focus Units Assay，FFU） 和磷酸盐缓冲液（PBS）的制备 ，其具体操作分别如下所示：
(1) 原代鸡胚成纤维细胞（CEC）的制备
选用产出一周以内、形态正常、蛋壳厚薄均匀一致、无裂纹、蛋白浓稠的种蛋，放入38±1℃、相对湿度60±20%的孵蛋器内进行孵育，用检卵灯观察是否为受精卵及其活力。选用9～11 日龄、鸡胚发育正常、可见清晰的血管及活动的鸡胚。用0.2%（m/v）的新洁尔灭溶液浸泡5分钟后捞出，气室向上置于蛋托上，然后用2%碘酊（m/v）和75%酒精（v/v）消毒后移入超净台内。用无菌镊子取出鸡胚，放入盛有1×Hanks溶液的平皿内。去除鸡胚的头、内脏，放入灭菌广口瓶内，用无菌剪剪切成1～3 mm3的组织块，按照每枚鸡胚5～8 ml的量加入预热至37℃的0.1%（m/v）胰酶溶液，置于37℃水浴箱内消化15～30分钟，加入50～300 ml细胞生长液（以199培养基为基础，添加终浓度为5～10%（v/v）的牛血清，199培养基可购自北京清大天一科技有限公司，型号为M199MD505），用无菌细胞吹打管吹打分散细胞得到细胞悬液，取1.0ml细胞悬液经10倍稀释后与等体积0.4%（m/v）台盼蓝混合均匀后，用血球计数板进行细胞计数，根据计数结果，将细胞密度调整为0.8～1.4×106个细胞/ml。
(2)PBS（pH7.4） 的制备
称取8.0g氯化钠，0.2g氯化钾，0.27g磷酸氢二钾，1.42g磷酸二氢钠，加入800ml注射用水充分搅拌混匀，然后加入38%的浓盐酸调pH至7.4，最后定容至1000ml。经121℃、15分钟灭菌后，室温保存备用。
(3) FFU 检测病毒滴度
①将待测病毒样品用PBS先进行10倍系列稀释，再进行3倍系列稀释。然后取50μl稀释后的病毒液接种50μl细胞密度为1×106个细胞/ml的BSR细胞（来源于中国疾病预防控制中心病毒病预防控制研究所）。混合均匀后置于37℃，5%CO2培养24小时。
②丙酮固定及染色
a.24小时后，倒弃上清液，用 PBS 洗涤一遍。
b.每孔加入50μl80%（v/v）冷丙酮，置于-20℃ 固定30分钟。
c.每孔加入50μl FITC标记的抗狂犬病病毒抗体（ Millipore 公司， Cat.NO.5100），置于37℃孵育30分钟。
d.用PBS洗涤三遍，甩干，每孔加入50μl 80％（v/v）甘油，直接置于荧光显微镜下观察每个稀释度的荧光灶数目。
e.取＞10个和＜10个荧光灶的相邻两孔稀释度的数据按照下列公式计算结果。
待测样品滴度（lgFFU/ml）=lg{[ （高稀释度的荧光平均数×3+低稀释度的荧光平均数）]/2×低稀释倍数×1000/50}
表1
样品编号 稀释度及荧光灶数目
孔1 孔2 孔1 孔2 孔1 孔2 孔1 孔2
1# 10-2×3 10-2×9 10-2×27 10-2×81
17 12 8 9
2# 10-3×3 10-3×9 10-3×27 10-3×81
25 29 8 6
备注：表格空白处表示荧光灶数目过多或者过少，无需记取。
根据表1中结果进行滴度的计算方法如下：
1#滴度 (lgFFU/ml）=lg{ [(8+9)/2]×3+(17+12)/2}/2×102×9×1000/50}=5.56
2# 滴度 (lgFFU/ml）=lg{ [(8+6)/2]×3+(25+29)/2}/2×103×27×1000/50}=7.11
实施例1
以CTN-1 V5株为母毒株，通过多条传代途径和工艺的比较筛选，发现按下列工艺路线传代获得的毒株能逐渐适应在鸡胚细胞中的生长，该狂犬病毒鸡胚细胞适应株被命名为CTNCEC25株。
步骤一：将CTN-1V5株在vero细胞上进行传代，以提高病毒滴度。
用PBS（pH7.4） 将RV CTN-1V5（来源于中国食品药品检定研究院，为CTN-1株vero细胞5代毒种） 做10倍系列稀释，然后按照1:100～1:1000的比例接种vero单层细胞（来源于中国食品药品检定研究院，121代），37 ℃吸附60分钟后补加细胞维持液（以199培养基为基础，添加终浓度10% （v/v）的牛血清，pH7.2～8.0） ，置于37 ℃、5%CO2培养箱内静置培养，培养4～6天收获病毒上清液，如此连续传10代，得到病毒滴度可达7.5lgLD50/ml以上的CTN-1V15株。
步骤二：将CTN-1V15株毒种在鸡胚中传1代，获得的RV CTN鸡胚一代病毒。
随后将获得的CTN-1V15株毒种经PBS（pH7.4）1:10～1:1000稀释后，取病毒稀释液经卵黄囊接种6～7日龄的SPF鸡胚，每胚0.5 ml，置于37～39℃、相对湿度40～80%的全自动孵蛋箱内进行孵育，逐日观察（24小时内死亡的接种胚记为非特异性死亡），孵育至72～144小时，收取濒临死亡但未死亡的胚胎，去除鸡胚的头后，将躯干研磨粉碎，并按照鸡胚重量加入病毒保护液（以199培养基为基础，添加终浓度20%（v/v）牛血清，pH7.2～8.0）制备成10%（m/v）的病毒悬液，然后2000 rpm（Revolutions Per Minute，rpm）、4℃离心10分钟，取上清按照1.0ml/支分装至专用冻存管，取样进行病毒滴度测定，如此传一代，获得RV CTN鸡胚一代病毒即CTNCE01，检测其滴度为5.2lgLD50/ml。
步骤三：将RV CTN鸡胚一代病毒在鸡胚成纤维细胞（CEC）中进行传代，使其逐渐适应CEC。
将步骤二获得的RV CTN鸡胚一代病毒（CTNCE01）用PBS（pH7.4）适宜稀释后（100～10-4），按照不同的接种比例（1:10～1:5×105）与前述制备好的CEC悬液（M0，细胞密度为0.8～1.4×106个细胞/ml）混合均匀，分装入细胞培养瓶内，置于35～37℃、5%CO2培养箱内培养。培养至细胞出现病变（主要表现为病变细胞聚集，圆缩，更快老化与脱落）收获病毒液，继续将病毒在CEC上按照上述培养条件进行传代。同时采用细胞荧光灶转化单位实验（Fluorescence Focus Units Assay，FFU，具体操作如下）对每一代毒种均取样测定病毒滴度，经过30～45次感染性和适应性试验后，在CEC中获得了满意的病毒滴度结果，经测序检验其5个结构蛋白基因一致，确认该毒株已为RV鸡胚细胞适应株，随将该毒种命名为RV CTNCEC25株，取33代毒株典藏于中国微生物菌种保藏管理委员会普通微生物中心。本发明中毒株的传代历史如图1所示。
将上述RV CTNCEC25株的33代制备为原始种子批毒种，将得到的原始种子批毒种按照MOI=0.001～0.05FFU/细胞的量接种于CEC，其它培养条件同上，连续传3代，得到主种子批毒种。主种子批毒种继续以制备主种子批毒种的培养条件在CEC上传4代，得到工作种子批毒种。
本发明得到的原始种子批毒种病毒滴度不低于6.5 lgFFU/ml，从原始种子得到的主种子批毒种和工作种子批毒种的滴度不低于7.0lgFFU/ml。
实施例2
本发明的RV CTNCEC25株的有关保藏信息如下：
分类命名: 狂犬病病毒（Rabies virus）。
保藏日期：2012年9月14日。
保藏单位名称：中国微生物菌种保藏管理委员会普通微生物中心。
保藏单位简称：CGMCC。
保藏单位地址：中国北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，邮政编码100101。
保藏编号：6510。
分类学:属于弹状病毒科（Rhabdoviridae）、狂犬病病毒属（Lyssaviruses）的一种新型毒株。
实施例3
上述RV CTNCEC25株有如下生物学特征：
1.毒株在原代鸡胚成纤维细胞（CEC）上的培养特性
将RV CTNCEC25株按照MOI=0.001～0.05 FFU/细胞的量接种于按照上述步骤制备的细胞密度为0.8～1.4×106个细胞/ml的CEC悬液，混合均匀后分装于细胞培养瓶或细胞工厂，置于33～36℃、5% CO2培养箱内培养，培养72小时后在倒置显微镜下（目镜10×，物镜20×）观察，发现细胞出现疏松、融合、空泡、胞质浓缩、胞浆中细胞器大量破坏甚至溶解的现象（图2），由此可见，该毒株能在原代CEC上形成明显的细胞病变。
2.毒株的感染性滴度
将RV CTNCEC25株按照MOI=0.005～0.01 FFU/细胞的量接种于按照上述步骤制备的细胞密度为0.8～1.4×106个细胞/ml的CEC悬液中，充分混合均匀后，置于33～36℃、5% CO2培养箱内培养，待80%以上的细胞出现病变后用一次性刻度吸管吸取0.5 ml病毒上清液，采用FFU法进行滴度测定。结果发现病毒接种细胞后72～120小时，其感染性滴度可达到7.0～8.0lgFFU/ml。
3.毒株对小鼠的脑内致病力
采用不同规格（11～13 g、18～20 g和28～30 g）的SPF级昆明白化小鼠各10只，以RV CTNCEC25株的100LD50的病毒量对上述小鼠进行脑内攻击，同时以其亲本RV CTN-1株同法处理作为对照，观察21天。结果发现经RV CTNCEC25株脑内攻击的小鼠，除11～13 g的小鼠有1只出现轻微的后肢麻痹症状（2天内症状消失）外，其余小鼠在观察期内皆未出现病症，而受亲本株RV CTN-1株脑内攻击的所有小鼠均出现典型的狂犬病病症且全部死亡。表明RV CTNCEC25株对成年小鼠的脑内致病力已大大减弱。
4.毒株对1～3日龄乳鼠的脑内致病力
以RV CTNCEC25株的100LD50的病毒量脑内攻击1～3日龄的SPF级的昆明乳鼠10只，同时以其亲本株CTN-1株同法处理作为对照，观察14天。结果发现受该毒株脑内攻击的乳鼠90%以上死亡，而其亲本CTN-1株攻击的乳鼠100%死亡。表明RV CTNCEC25株对乳鼠仍然具有较强的脑内致病力和致死性。
5.毒株对成年小鼠的皮下、肌肉和腹腔致病力
以最高剂量均为106.5LD50的RV CTNCEC25株和RV CTN-1株病毒液，分别通过皮下、腹腔、肌肉途径注射不同规格（11～13g、18～20g和28～30g）的SPF级昆明白化小鼠，不同途径、不同规格各10只小鼠，观察21天。结果发现RV CTNCEC25株通过皮下、腹腔或者肌肉注射小鼠，小鼠均不发病。而其亲本株RV CTN-1株注射组均有数量不等的小鼠出现狂犬病病症和死亡，具体结果见表2。表明与其亲本株RV CTN-1株相比，RV CTNCEC25株已完全丧失了对成年小鼠的皮下、肌肉和腹腔的致病力。
表2
小鼠规格（g） 注射途径 注射数量（只） 3天内非特异性死亡数量（只） 21天内死亡数目(只)
11～13 CTNCEC25株 皮下 10 0 0
肌肉 10 0 0
腹腔 10 0 0
CTN-1株 皮下 10 0 6
肌肉 10 0 4
腹腔 10 0 0
18～20 CTNCEC25株 皮下 10 0 0
肌肉 10 0 0
腹腔 10 0 0
CTN-1株 皮下 10 0 5
肌肉 10 0 6
腹腔 10 1 0
28～30 CTNCEC25株 皮下 10 0 0
肌肉 10 0 0
腹腔 10 0 0
CTN-1株 皮下 10 0 1
肌肉 10 0 1
腹腔 10 0 0
6.毒株的特异性鉴别
采用FFU法做中和试验，鉴定RV CTNCEC25株毒种的特异性。具体方法如下所示：
(1)将RV CTNCEC25株毒种用PBS先做10倍系列稀释，再做3倍系列稀释，然后取适宜稀释度的病毒稀释液（一般试验组取3-1×10-1～3-4×10-1稀释度，对照组取3-1×10-4～3-4×10-3稀释度）分别与稀释度10-2狂犬病病毒特异性免疫血清（来源于武汉生物制品研究所，效价200IU/ml）（试验组）和稀释度10-2阴性马血清（来源于广州蕊特生物科技有限公司）（对照组）等量混合，置于37℃水浴中和60分钟。水浴结束后取50μl中和后的病毒液接种50μl细胞密度为1×106个细胞/ml的BSR细胞（来源于中国疾病预防控制中心病毒病预防控制研究所，25代）。混合均匀后置于37℃，5%CO2培养24小时。最后如前所示进行丙酮固定及染色。
取＞10个和＜10个荧光灶的相邻两孔稀释度的数据按照下列公式计算中和指数：
中和指数=对照组滴度（FFU/ml）/试验组滴度（FFU/ml）
具体测定结果如表3所示：
表3
代次 组别 病毒稀释倍数及荧光灶数 滴度（FFU/ml）
33代 试验组 10 30 2100
13,14 2,3
对照组 10000 30000 2.9×106
23,23 3,1
33代中和指数=2.9×106/2100 =1381
36代 试验组 30 90 10050
16,18 6,5
对照组 27×10000 81×10000 5.87×107
28,17 6,8
36代中和指数=5.87×107/10050=5841
40代 试验组 90 270 22500
13,13 4,4
对照组 81×10000
13,17 ＞1.22×108
40代中和指数＞1.22×108/22500=5422
结果发现毒株中和指数可达1000以上，符合2010年版《中国药典》“中和指数不低于500”的规定，表明该毒株为RV毒株。
7.无菌检查
依据2010年版《中国药典》进行检查，结果符合规定。
8.支原体检查
依据2010年版《中国药典》进行检查，结果符合规定。
9.外源因子检查
依据2010年版《中国药典》进行检查，结果符合规定。
10.免疫原性检查
以MOI=0.001～0.05FFU/细胞的接种量将RV CTNCEC25株接种于按照上述步骤制备好的细胞密度为0.8～1.4×106个细胞/ml的CEC悬液中，混合均匀，置于33～36℃孵育4～6天，待80%以上的细胞出现病变时，收获病毒上清液。病毒上清液经Ф0.60μm针孔式过滤器（MIillipore公司）粗滤后，缓慢加入β-丙内酯，使β-丙内酯与粗滤后病毒液的最终比例达到1:4000（v/v），充分搅拌混匀后置于2～8℃进行孵育，需要孵育至少24小时以充分灭活病毒感染性而不丧失病毒的抗原性。24小时后将经β-丙内酯灭活的病毒液放入37℃的水浴箱内水浴2小时，使病毒液中残余的β-丙内酯充分水解，即得到试验用原疫苗。将上述步骤获得的原疫苗，按照2010年版《中国药典》规程检查其免疫保护性。具体实验结果见表4。试验表明该毒株的保护指数可达50000以上，远远大于2010年版《中国药典》 “中和指数不低于500” 的规定，表明该病毒具有良好的免疫保护性。
表4
代次 保护指数 备注
33代 53703 原始种子批毒种
36代 72444 主种子批毒种
40代 61660 工作种子批毒种
11.基因序列测定与分析
采用分子生物学技术，根据NCBI网站Genbank公布的RV CTN-1株的序列，设计RV CTNCEC25株全基因组特异性测序引物，经英潍捷基生物技术有限公司合成，具体信息见图4。利用RT-PCR的方法扩增该毒株的全基因组序列，进行序列测定和拼接（由英潍捷基生物技术有限公司完成），使用DNAStar软件中的MegAlign工具对基因组序列进行分析，并根据测定的毒株核苷酸序列获得了本发明毒株的5个已知结构蛋白的氨基酸序列。测定结果表明，该毒株核苷酸全长为11924bp（Genbank accession： KJ466147），其中N基因含1425个核苷酸，由450个氨基酸组成；P基因含989个核苷酸，由297个氨基酸组成；M基因含803个核苷酸，由202个氨基酸组成；G基因含2067个核苷酸，由524个氨基酸组成；L蛋白含6474个核苷酸，由2128个氨基酸组成。
RV CTNCEC25株的全基因序列如下：
Rabies virus strain CTNCEC25，complete genome
GenBank： 11924bp cDNA
TACGCTTAACAACCAAATCAAATAAGAAACAGACAGTGTCATTTGCAAAACAAAAATGTAACACCCCTACAATGGATGCCGACAAGATTGTATTCAGAGTCAATAATCAGGTGGTCTCCTtGAAGCCCGAGATTATtGTGGATCaATATGAATACAAATATCCGGCCATCAAAGACTTGAAGAAAcCCAGTATCACCCTAGGGAAAGCTCCCGACTTAAACAAGGCATACAAGTCAGTCCTGTCAGGCATGAATGCTGcCAAGCTGGATCCGGATGATGTATGTTCTTACTTAGCAGCTGCAATGCAATTCTTTGAGGGGACATGCCCTGAAGACTGGAATAGCTACGGGATCTTGATTGCACGAAAAGGAGACAAAATCACCCCTGATTCTCTTGTAGATATAAAGCGTACTGACGTAGAAGGGAACTGGgCTCtGACGGGAGGTATGGAGTTGACGAGAGACCCCACTGTGTCTGAGCATGCGTCTTTAGTTGGTCTTCTCTTGAGTCTTTATAGGTTGAGCAAAATATCGGGACAAAACACAGGCAACTACAAGACAAACATTGCGGACAGGATAGAGCAGATTTTCGAGACTGCCCCTTTCGTCAAGATCGTAGAACACCATACTCTGATGACAACTCACAAGATGTGTGCTAACTGGAGTACCATACCGAACTTCAGGTTCTTAGCCGGAACCTACGATATGTTTTTCTCCCGGATTGAACATCTGTATTCAGCTATTAGAGTGGGTACAGTCGTCACTGCTTATGAAGACTGCTCAGGGCTAGTATCGTTTACAGGGTTCATAAAGCAGATAAATCTCACTGCAAAGGAGGCAATACTATATTTCTTCCATAAAAACTTTGAGGAAGAGATAAGAAGAATGTTTGAACCAGGGCAAGAGACTGCTGTTCCTCACTCTTATTTCATCCACTTCCGTTCATTAGGCCTGAGTGGAAAGTCTCCATATTCATCAAATGCAGTTGGTCACGTGTTCAATCTCATTCACTTTGTTGGATGCTATATGGGTCAAGTCAGATCTCTTAATGCAACGGTTATTGCCGCATGTGCACCCCATGAGATGTCTGTTCTGGGAGGCTACTTGGGAGAGGAGTTTTTTGGAAAGGGGACGTTTGAAAGAAGATTCTTCAGAGATGAGAAAGAACTTCAAGAATATGAGGCGGCTGAGTTAACCAAGACTGACTTAGCACTGGCAGACGATGGAACCGTCAACTCTGATGATGAAGACTATTTCTCCGGTGAAACCAGGAGTCCGGAAGCTGTCTATACTCGAATCATGATGAACGGAGGTCGACTAAAGAGATCGCACATAAGGAGATATGTATCTGTCAGCTCCAATCATCAAGCCCGTCCAAACTCATTTGCCGAGTTTCTAAACAAGACGTACTCTAGTGACTCGTAAAGAATTGAACAACAAGATTGAAAAGAACCTACAAAGTGTGTATATCCTTCATGAAAAAAACTAACACCCCTCCTTTTGAACCATCTCAACGATGAGCAAGATCTTCGTCAATCCGAGTGCAATCAGAGCCGGTTTGGCTGACCTCGAGATGGCTGAAGAGACTGTTGATCTAATCAACAGAAACATAGAAGATAATCAGGCCCATCTCCAGGGAGAACCCATAGAAGTGGACAATCTCCCTGAGGACATGAGGCGGCTTCAGTTAGATGATGGGAAGCCGTCGGACCTCAGTGAGATGGCCCCGGCGGGAGAGAGCAAGTATCGGGAGGACTTTCAGATGGATGAAGGGGAGGATCCTGGCCTCTTGTTCCAGTCATACCTGGAAAATGTTGGAGTCCAAATAGTCAGACAAATGAGGTCTGGAGAGAGATTCCTCAAGATTTGGTCACAGACTGTAGAGGAAATAGTCTCCTATGTCATGACTAACTTCCCTAACCCATCGGGAAGATCCTCGGAGGACAAATCTACCCAGACTGCTGGGAGAGAACTTAAGAAGGAAGCTCCATCGGCTTCTTCTCAGAGAGAGAACCAATCCTCAAAAGCCCGAATGGCAGCTCAAACTGCCTCTGGTCCTCCTGCTCTCGAATGGTCTGCGACCAACGACGAAGATGATCTGTCTGTAGAGGCGGAGATTGCTCACCAGATTGCTGAGAGCTTCTCCAAGAAATACAAATTCCCTTCTAGGTCATCAGGAATATTCTTGTACAACTTTGAGCAGTTGAAAATGAACCTTGACGACATAGTCAAGGAGTCAAAAAATGTCCCCGGTGTGACCCGTTTAGCTCATGATGGATCTAAACTCCCTCTAAGATGTGTACTGGGGTGGGTTGCTCTGGCTAACTCTAAGAAGTTCCAGCTATTGGTCGAGCCAGACAAGTTGAATAAAGTTATGCAGGATGATCTAAATCGTTATACATCTTGCTGACAATTTTTAAACTCAGGCCTACCAGACTATAGGGCCTGAGCTGTTCTGGACTCAACATGGaAAAAACAGGCAACACCACTGATAAAATGAACTTTCTACGCAAGATAGTGAAAAACTGTAGGGATGAGGACACCCAGAAGCCCCCCCCTGTATCGGCTCCTCCAGATGATGATGACTTGTGGCTGCCCCCTCCTGAATATGTCCCGCTGAAAGAACTCACGGGCAAGAAGAACATGAGGAACTTCTGCATCAATGGTGAGGTCAAGGTGTGCAGTCCAAACGGCTATTCATTCAGGATCTTACGGCATATTCTAAGATCATTTGATGAGATATATTCTGGAAATCACAGAATGATTGGATTAGTCAAGGTAGTCATTGGGCGTGCTTTATCAGGGGCTCCGGTCCCTGAGGGCATGAACTGGGTGTACAAATTGAGGAGAACTCTTATCTTCCAATGGGCTGATTCTAGAGGTCCCCTCGAGGGGGAGGAGTTAGAGTACTCTCAAGAGATCACCTGGGATGACGATACTGAGTTTGTCGGATTGCAGATAAGAGTGAGTGCAAGACAATGTCATATCCAGGGTAGGGTATGGTGTATCAACATGAATTCTAGAACATGTCAACTATGGTCTGACATGTCTCTTCAGACACAAAGGTTGGAGGAGGACAAGGACTCTTCCCTGCTCCTAGAATAGTCAAATTATATCTCACAGACTTATCACTTGTTTACCTCTGGAGGAGAGAACATACGGGCTTAACTCCAACCTTTGGGAGCAATAGAACAAAAATATGTTATGGTGCCATTAAACCGCTGCATTTTATCAGAATCAGATTAATTACCTTTACATTTTGAGCCTCTTGGATGTGAAAAAAACTTTTAACATCCCTCAAAAGACTTAAGGAAAGATGATTCCTCAAGCTCTGTTGTTTGTACCTCTTCTGGTTTTTCCATTGTGTTTCGGGAAATTCCCCATTTACACGATACCAGACAAACTCGGCCCCTGGAGTCCCATCGATATACATCACCTCAGCTGTCCGAACAATCTGGTTGTGGAGGACGAAGGATGTACCAATCTGTCAGGATTCTCATACATGGAGCTTAAAGTAGGATATATTTCGGCCATAAAGGTGAACGGGTTCACTTGTACGGGTGTGGTAACGGAAGCAGAAACCTACACTAACTTTGTCGGTTATGTCACCACCACGTTTAAGAGAAAGCACTTCCGACCAACACCGGATGCATGCAGATCAGCATACAATTGGAAGATGGCAGGTGACCCCAGATATGAAGAGTCTCTGCACAATCCCTATCCTGATTATCATTGGCTCCGGACTGTAAAAACCACCAAAGAGTCTGTTGTTATCATATCTCCAAGTGTGGCAGACTTAGACCCGTACGATGAATCACTTCATTCGAGAGTTTTTCCTAGAGGAAAATGCTCAGGAATAACGGTGTCTTCTGCCTACTGCTCTACCAACCATGATTATACCATCTGGATGCCTGAAAATCCTAGACTGGGGACCTCTTGTGATAtTTTCACCAACAGCAGAGGGAAGAGAGCATCCAAAGGGAGCAAGACCTGTGGATTTGTGGATGAGAGAGGCTTGTACAAATCTCTAAAAGGAGCATGCAAACTGAAGCTGTGTGGAGTTCTTGGACTTAGGCTTATGGACGGAACCTGGGTCGCGATTCAGACATCAAACGAGACCAAGTGGTGCCCTCCTGATCAACTAGTGAATCTACATGACTTTCATTCAGATGAGATTGAACATCTTGTTGTGGAGGAGTTGGTTAAGAAGAGGGAGGAGTGTCTAGATGCACTGGAGTCCATCATGACCACCAAGTCCGTGAGTTTCAGACGTCTCAGTCACTTGAGGAAGCTTGTGCCTGGATTTGGAAAAGCATACACCATATTCAACAAGACCTTAATGGAGGCTGATGCTCACTACAAATCGGTCCAAACTTGGAATGAGATCATCCCCTCGAAAGGGTGTTTAAGAGTCGGGGGGAGATGTCATCCTCATGTGAACGGAGTATTTTTCAATGGTATCATCCTAGGCCCTGACGGCCATGTCTTAATCCCGGAAATGCAGTCATCCCTCCTCCAGCAGCATATGGAGTTGTTGAAATCCTCGGTCATCCCCTTAATGCATCCCTTGGCAGATCCATCAACGGTTTTTAAAGATGGTGACGAGGTGGAGGATTTTGTTGAGGTTCACCTTCAGGATGTGCATAAGCAGGTCTCAGGGGTTGATCTCGGTCTCCCAAACTGGGGGAAGGATGTGTTGATGGGCGCAGGCGTTTTGACGGCACTGATGTTGATGATTTTCCTAATGACGTGTTGCCGAAGGACTAATAGAGCAGAATCAATACAACACAGTCTTGGAGAGACAGGGAGGAAAGTGTCGGTGACCCCCCAAAGCGGGAGGGTCATATCTTCATGGGAGTCATATAAAAGCGGAGGTGAGACCAAGCTGTAAAGGCAGGTCATCTCCTTCATATTTCAAGTCCCGAGGATCCCATCCCCTTGAGGTGAAGGGGACATCTCTGGATTCAATGGTCCTCTTTGGACTCCTCACAACAGGGTAGATTCAAGAGTCTTGAGACCTCCATTAATCATCTCAATCGATCAGACATGGTCATGTAGATTCTTATAACACAAGAAATCTTCTAGCAGTTCCAGTGACCAACGGTGCTTTTACCCTCCAAGAACAGATACCAAAGGTGGTGGACAAGCCTAGAGATATCTCAGATGATGTTGTGCTTCAACACAGACAGAGATTGTAGTGAGTCCCCCGATATTAGACTGGGTAAGAGTCTGTTAAGAAAGAATACTTGCATCCTATGAAGGACATAAGCAATAGATCACCATCATCTTGCATCTCAGCAAAGTGTGCATAATTATAAAGGGCTGGGTCATTCATGATTTTCAGTCGAGAAAAAAACTATAGAGCTGAAGGACAACTAGCAACACTTCTCATCTTGAAACTCTCACCATGATGATTGATCCAGGAGAGGTTTATGATGACCCGGTTGATCCGATTGAGTCAGAGGCCGAGCCCAGAGGGACTCCCACTGTCCCCAACATCTTGAGGAACTCTGACTATAATCTCAACTCTCCTCTAATAGAGGATCCTGCCAAATTAATGTTAGAATGGTTGAAGACAGGAAATAGACCTTACCGGATGACTTTGACAGACAACAGCTCTAGGTCTTACAAAGTCCTGAAAGATTACTTCAAGAAGTTGGATCTGGGTTCCCTCAAAGTGGGTGGAACTGCAGCCCAGTCAATGATCTCCCTTTGGTTGTATGGTGCTCACTCTGAATCAAACAGGAGCCGGAAATGTATAACTGACTTGGCCCATTTTTATTCCAAGTCCTCCCCCATAGAGAAGCTGTTGAATTGCACACTTGGAAATAGAGGGCTAAGAATCCCTCCAGAAGGGGTGTTAAGTTGCCTTGAAAGAGTTGACTATGACAAGGCATTTGGAAGATATCTCTCCAATACGTATTCCTCTTATCTGTTCTTCCACGTGATTACCCTGTACATGAATGCCCTAGACTGGGATGAAGAGAAGACCATCTTAGCTTTATGGAAAGACTTGACGTCAGTTGATGTTGGGAAAGACTTAGTCAAGTTCAAAGATCAAATATGGGGACTACTAATTGTGACGAAGGACTTTGTGTACTCTCAGAGTTCTAATTGTCTTTTTGACAGGAACTATACACTTATGCTGAAAGACCTTTTTTTGTCTCGTTTCAACTCCCTAATGATTCTGCTTTCTCCTCCCGAACCCCGGTATTCAGACGACCTGATATCCCAACTTTGCCAACTATACATTGCAGGAGATCAAGTATTGTCCATGTGTGGAAATTCGGGTTATGAAGTCATAAAGATACTGGAACCCTATGTCGTGAACAGTTTAGTCCAGAGGGCAGAGAAGTTTAGGCCCCTCATCCACTCTTTGGGGGACTTTCCCGTTTTTATAAAGGACAAAGTGAATCAACTCGAGGGGACATTCGGTCCCAGTGCAAAAAGGTTTTTTCGGGTTTTGGATCAATTCGACAACATACATGACCTAGTCTTTGTGTATGGCTGTTATAGACATTGGGGGCATCCTTATATAGATTATAGGAAGGGTCTGTCAAAGCTATACGATCAAGTTCATATCAAAAAGGTGATAGATAAATCCTATCAGGAGTGCTTGGCGAGCGACCTGGCCAGGAGGATCCTTAGGTGGGGATTTGACAAGTACTCTAAATGGTATATTGACTCACGATTGCTCCCCAGAGACCACCCCTTGACTCCTTATATAAAAACTCAGACATGGCCACCCAAACATGTAGTAGATTTGGTAGGGGACACATGGCACAGGCTACCAATCACACAGATATTTGAGATCCCTGAGTCAATGGACCCTTCAGAAATACTAGATGACAAGTCGCACTCTTTCACCAGAACAAGACTAGCTTCCTGGCTGTCAGAAAATAGAGGGGGGCCTGTTCCTAGTGAGAAGGTCATTATCACAGCTCTGTCCAAGCCGCCTGTCAATCCTAGAGAGTTTTTAAAATCTATCGACCTCGGAGGATTACCCGATGAAGACTTGATAATTGGCCTCAAGCCAAAGGAGAGGGAGCTGAAGATCGAAGGTCGATTTTTCGCCTTAATGTCTTGGAATCTGAGACTGTACTTCGTTATCACCGAAAAGCTCTTAGCTAATTATATCTTGCCACTCTTTGATGCGCTGACTATGACAGACAACCTGAACAAGGTGTTTAAAAAGTTGATTGATCGAGTAACCGGGCAAGGGCTCTTGGACTACTCACGGGTTACATACGCTTTTCACCTAGACTATGAGAAGTGGAACAATCATCAACGGTCAGAGTCGACAGAAGATGTATTTTCCGTCCTAGATCAGGTGTTTGGATTAAAGAGGGTGTTCTCGAGGACTCATGAGTTTTTCCAGAAGTCTTGGATCTATTATTCGGACAGATCAGACCTCATCGGGCTATGGGAGGACCAAATACACTGCTTAGATATGTCTAACGGCCCGACCTGCTGGAATGGACAAGATGGCGGGCTGGAAGGCTTACGGCAGAAAGGCTGGAGTCTGGTCAGCCTATTGATGATAGACCGAGAGTCTCAGACTAGGAACACAAGGACCAAGATACTGGCGCAAGGAGACAACCAGGTTCTATGTCCGACATATATGTTGTCTCCTGGGCTGTCGAGAGAAGGGCTTCTTTATGAACTGGAGAGCATATCAAGAAATGCCCTATCAATATACCGAGCTATAGAGGAAGGAGCATCCAAGCTGGGGTTGATTATCAAGAAGGAAGAGACCATGTGCAGCTATGACTTTCTCATCTATGGGAAAACTCCATTGTTTCGAGGTAACATCTTGGTGCCGGAGTCAAAAAGATGGGCCAGAGTCTCTTGCATCTCGAACGACCAAATAGTTAACCTTGCCAATATAATGTCAACAGTATCCACCAACGCCTTAACTGTGGCACAACACTCTCAATCTTTGATCAAGCCTATGAGAGACTTTTTGCTTATGTCAGTGCAGGCAGTCTTTCATTACTTGCTGTTTAGCCCCATCTTAAAAGGCAGAGTCTACAAAATTCTAAGTGCTGAGGGGGACAATTTTCTCCTAGCGATGTCAAGAATAATCTATCTGGACCCTTCCTTAGGGGGAGTGTCTGGAATGTCTTTGGGGAGATTTCACATACGCCAGTTCTCAGATCCTGTCTCAGAAGGGTTGTCCTTCTGGAGAGAGATTTGGCTGAGCTCTCACGAGTCTTGGATTCACGCCTTGTGTCAGGAGGCAGGGAACCCCGACCTTGGGGAGAGAACACTCGAGAGCTTCACTCGGCTTCTCGAGGATCCTACTACTTTAAATATCAAGGGAGGGGCAAGCCCTACTATCTTACTCAAGGATGCGATCAGGAAAGCCCTGTATGACGAAGTGGATAAGGTGGAGAACTCTGAGTTCAGAGAGGCAATCCTCTTGTCCAAAACCCATAGGGATAACTTTATACTTTTCTTGAAATCTGTTGAACCTCTGTTTCCCCGATTTCTTAGTGAGCTTTTCAGTTCATCCTTCTTGGGGATTCCGGAGTCAATCATCGGATTGATACAAAACTCTCGGACGATCAGAAGGCAGTTTAGAAAAAGCCTCTCAAGAACTTTGGAGGAGTCCTTTTACAACTCAGAGATCCATGGAATCAACCGGATGACTCAGACACCTCAGCGAGTTGGGAGGGTGTGGCCATGCTCCTCGGAGAGGGCAGATCTTTTGAGGGAAATCTCTTGGGGGAGGAAAGTAGTAGGTACAACAGTTCCTCACCCTTCTGAGATGTTGGGACTGCTTCCTAAGTCCTCTATCTCTTGTACTTGTGGAGCAACAGGAGGAGGGAATCCTAGAGTTTCAGTGTCTGTGCTTCCGTCCTTTGATCAGTCATCCTTTTCAAGAGGCCCTCTAAAGGGATACCTGGGATCATCTACCTCCATGTCAACCCAGCTATTCCATGCTTGGGAGAAGGTCACCAATGTTCATGTAGTAAAAAGGGCCCTCTCACTAAAAGAATCTATAAACTGGTTCATCACAAGAAACTCGAATTTGGCTCAGACTCTGATTAGAAACATCATGTCTCTGACAGGCCCTGATTTTCCTCTAGAAGAGGCCCCTGTGTTCAAAAGAACCGGGTCAGCATTGCATAGGTTCAAGTCCGCCAGATACAGTGAAGGGGGATATTCTTCTGTATGCCCCAATCTCCTCTCTCATATTTCCGTGAGTACAGACACCATGTCTGATTTGACCCAGGACGGGAAGAACTATGATTTCATGTTCCAGCCCTTGATGCTTTATGCGCAGACATGGACATCCGAATTGGTACAAAGAGACACAAGGCTGAAGGACTCTACGTTCCACTGGCACCTCCGATGTAACAGGTGTGTAAGGCCAATAGACGATATCACACTGGAGACCTCTCAGGTCTTCGAGTTTCCGGACGTGTCAAAAAGAATATCTAGGATGGTTTCTGGAGCTGTGCCTCACTTCCAAAAACTCCCTGATATCCGCCTGAAGCCTGGGGATTTTGAATCTTTAAGTGGTAGGGAAAAATCCCGTCACATAGGTTCAGCTCAGGGGCTTCTATATTCGATCTTGGTCGCGATACATGACTCAGGATACAATGACGGAACTATCTTCCCTGTCAACATATATGGCAAGGTCTCCCCTAGAGACTATTTAAGGGGGCTTGCAAGAGGAGTTCTAATAGGGTCGTCCATATGCTTCTTGACCAGAATGACTAACATCAACATTTATAGGCCTCTAGAGTTGATCTCAGGTGTGATCTCATATATCCTCTtGAGGCTGGATAATCACCCATCTTTGTACATaATGCTCAGAGAACCATCTCTTAGAGGAGAGATATTTTCTATACCCCAGAAAATTCCGGCTGCTTACCCTACCACGATGAAGGAGGGTAATAGATCTATCTTGTGTTACCTCCAACATGTGCTGCGCTATGAGCGAGAGGTTATTACAGCATCTCCAGAGAATGACTGGTTGTGGATCTTTTCGGACTTTAGAAGCTCCAAGATGACATACTTAACTCTCATCACTTATCAGTCTCATCTTCTACTTCAGAGGGTTGAAAGAAATCTGTCGAAGAGTATGAGAGCTAACCTGCGACAGATGAGTTCCTTGATGAGGCAGGTACTAGGAGGGCATGGGGAGGACACCTTAGAATCGGATGATGACATTCAAAGGCTATTAAAGGACTCTCTGCGTAAAACGAGGTGGGTGGATCAAGAAGTGCGCCATGCAGCCAGAACTATGACGGGGGATTACAGCCCCAATAAGAAGATGTCACGCAAGGCGGGATGCTCTGAGTGGGTTTGTTCCGCTCAGCAGGTTGCAGTCTCCACCTCGGCTAACCCAGCCCCTGTTTCAGAGCTTGACATAAGGGCTCTCTCCAAGAGGTTTCAGAACCCTCTCATCTCGGGATTAAGAGTGGTTCAGTGGGCAACTGGTGCCCATTATAAACTCAAGCCCATTCTGGATGATCTCAATGTCTTTCCATCCTTATGCCTTGTGGTTGGGGACGGATCAGGAGGAATATcGCGAGCAGTACTCAACATGTTTCCAGATGCCAAGCTTGTATTCAACAGCTTGTTGGAGGTGAATGACCTGATGGCATCCGGAACACATCCGTTGCCTCCCTCAGCCATCATGAGTGGAGGAGATGATATTGTCTCCAGAGTCATAGACTTCGACTCAATCTGGGAAAAGCCATCAGATCTCAGGAACTTGACTACATGGAAGTACTTCCAGTCAGTCCAGAAAAGGGTGAACATGTCTTATGACCTCATCATTTGTGATGCAGAAGTCACTGACATAGCATCAATCAATCGGATAACACTGTTGATGTCCGACTTCGCATTGTCAATAGACGGCCCACTGTATCTGGTCTTCAAAACTTATGGAACTATGCTGGTAAATCCAGAATATAGAGCAATTCAACACCTGTCAAGGGCGTTTCCTTCAGTCACAGGCTTTATAACCCAGATGACCTCATCCTTCTCATCCGAACTATACCTCCGATTTTCCAAGCGAGGAAAATTCTTCCGAGATGCCGAGTACTTAACTTCTTCTACCCTTCGAGAGATGAGCCTCGTCTTGTTCAACTGTAGTAGCCCTAAGAGTGAGATGCAGAGGGCTCGCTCTTTGAATTACCAAGATCTTGTAAGAGGATTTCCAGAGGAGATTATATCCAATCCTTACAATGAAATGATCATAACCTTGATTGACAGTGATGTGGAATCTTTCCTGGTTCATAAGATGGTAGATGACCTAGAATTACAAAGAGGGACTTTATCTAAGATGGCTGTCATTATAGCCATTATGATAGTTTTCTCCAATAGAGTTTTCAATGTTTCTAAACCATTGACCGACCCTTTGTTCTATCCACCATCGGACCCCAAAATCCTGAGGCACTTCAACATATGCTGCAGTACAATGATGTATCTGGCAACGGCTCTAGGTGATGTCCCCAGCTTTGCTAGGCTTCATGACTTGTACAATAGGCCTATAACTTATTACTTTAAGAAGCAGGTTATTCGAGGGAGTATTTATCTGTCTTGGAGTTGGTCTGACGATACCTCAGTGTTCAAGAGGGTGGCCTGCAACTCTAGTTTGAGTCTGTCGTCTCATTGGATCAGGCTTGTTTACAAGATAGTGAAGACCACCAGACTTGTCGGGAGTATTGAAGACTTATCTGGAGAGGTGGTGAGACATCTTCAAGGGTATAACAGGTGGATTACCCTCGAGGACATCAGATCTAGATCATCTCTATTAGACTACAGCTGCTTATGAACTGAACATTATTGGGGCTTGTAAATACCGAAACTCTTAGGCGATGCACCTTGAAAAAAACAAGATCTCAAATCAGAACCTCTGGTTGCTTGATTATTTTTTTCATCTTTATGGTCTTTTTGTTAAGCGTA
RV CTNCEC25株5个结构蛋白的氨基酸序列如下：
gene 60..1484
/gene="N"
mRNA 60..1484
/gene="N"
/product="nucleoprotein"
CDS 72..1424
/gene="N"
/codon_start=1
/product="nucleoprotein"
/translation="MDADKIVFRVNNQVVSLKPEIIVDQYEYKYPAIKDLKKPSITLGKAPDLNKAYKSVLSGMNAAKLDPDDVCSYLAAAMQFFEGTCPEDWNSYGILIARKGDKITPDSLVDIKRTDVEGNWALTGGMELTRDPTVSEHASLVGLLLSLYRLSKISGQNTGNYKTNIADRIEQIFETAPFVKIVEHHTLMTTHKMCANWSTIPNFRFLAGTYDMFFSHLYSAIRVGTVVTAYEDCSGLVSFTGFIKQINLTAKEAILYFFHKNFEEEIRRMFEPGRIEQETAVPHSYFIHFRSLGLSGKSPYSSNAVGHVFNLIHFVGCYMGQVRSLNATVIAACAPHEMSVLGGYLGEEFFGKGTFERRFFRDEKELQEYEAAELTKTDLALADDGTVNSDDEDYFSGETRSPEAVYTRIMMNGGRLKRSHIRRYVSVSSNHQARPNSFAEFLNKMYSSDS"
gene 1487..2475
/gene="P"
mRNA 1487..2475
/gene="P"
/product="phosphoprotein"
CDS 1516..2409
/gene="P"
/codon_start=1
/product="phosphoprotein"
/translation="MSKIFVNPSAIRAGLADLEMAEETVDLINRNIEDNQAHLQGEPIEVDNLPEDMRRLQLDDGKPSDLSEMAPAGESKYREDFQMDEGEDPGLLFQSYLENVGVQIVRQMRSGERFLKIWSQTVEEIVSYVMTNFPNPSGRSSEDKSTQTAGRELKKEAPSASSQRENQSSKARMAAQTASGPPALEWSATNDEDDLSVEAEIAHQIAESFSKKYKFPSRSSGIFLYNFEQLKMNLDDIVKESKNVPGVTRLAHDGSKLPLRCVLGWVALANSKKFQL LVEPDKLNKVMQDDLNRYTSC"
gene 2482..3284
/gene="M"
mRNA 2482..3284
/gene="M"
/product="matrix protein"
CDS 2497..3105
/gene="M"
/codon_start=1
/product="matrix protein"
/translation="MNFLRKIVKNCRDEDTQKPPPVSAPPDDDDLWLPPPEYVPLKELTGKKNMRNFCINGEVKVCSPNGYSFRILRHILRSFDEIYSGNHRMIGLVKVVIGRALSGAPVPEGMNWVYKLRRTLIFQWADSRGPLEGEELEYSQEITWDDDTEFVGLQIRVSARQCHIQGRVWCINMNSRTCQLWSDMSLQTQRLEEDKDSSLLLE"
gene 3290..5356
/gene="G"
mRNA 3290..5356
/gene="G"
/product="glycoprotein"
CDS 3317..4891
/gene="G"
/codon_start=1
/product="glycoprotein"
/translation="MIPQALLFVPLLVFPLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRSAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKESVVIISPSVADLDPYDESLHSRVFPRGKCSGITVSSAYCSTNHDYTIWMPENPRLGTSCDIFTNSRGKRASKGSKTCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAIQTSNETKWCPPDQLVNLHDFHSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVQTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGHVLIPEMQSSLLQQHMELLKSSVIPLMHPLADPSTVFKDGDEVEDFVEVHLQDVHKQVSGVDLGLPNWGKDVLMGAGVLTALMLMIFLMTCCRRTNRAESIQHSLGETGRKVSVTPQSGRVISSWESYKSGGETKL"
gene 5381..11854
/gene="L"
mRNA 5381..11854
/gene="L"
/product="large protein"
CDS 5408..11794
/gene="L"
/codon_start=1
/product="large protein"
/translation="MMIDPGEVYDDPVDPIESEAEPRGTPTVPNILRNSDYNLNSPLIEDPAKLMLEWLKTGNRPYRMTLTDNSSRSYKVLKDYFKKLDLGSLKVGGTAAQSMISLWLYGAHSESNRSRKCITDLAHFYSKSSPIEKLLNCTLGNRGLRIPPEGVLSCLERVDYDKAFGRYLSNTYSSYLFFHVITLYMNALDWDEEKTILALWKDLTSVDVGKDLVKFKDQIWGLLIVTKDFVYSQSSNCLFDRNYTLMLKDLFLSRFNSLMILLSPPEPRYSDDLISQLCQLYIAGDQVLSMCGNSGYEVIKILEPYVVNSLVQRAEKFRPLIHSLGDFPVFIKDKVNQLEGTFGPSAKRFFRVLDQFDNIHDLVFVYGCYRHWGHPYIDYRKGLSKLYDQVHIKKVIDKSYQECLASDLARRILRWGFDKYSKWYIDSRLLPRDHPLTPYIKTQTWPPKHVVDLVGDTWHRLPITQIFEIPESMDPSEILDDKSHSFTRTRLASWLSENRGGPVPSEKVIITALSKPPVNPREFLKSIDLGGLPDEDLIIGLKPKERELKIEGRFFALMSWNLRLYFVITEKLLANYILPLFDALTMTDNLNKVFKKLIDRVTGQGLLDYSRVTYAFHLDYEKWNNHQRSESTEDVFSVLDQVFGLKRVFSRTHEFFQKSWIYYSDRSDLIGLWEDQIHCLDMSNGPTCWNGQDGGLEGLRQKGWSLVSLLMIDRESQTRNTRTKILAQGDNQVLCPTYMLSPGLSREGLLYELESISRNALSIYRAIEEGASKLGLIIKKEETMCSYDFLIYGKTPLFRGNILVPESKRWARVSCISNDQIVNLANIMSTVSTNALTVAQHSQSLIKPMRDFLLMSVQAVFHYLLFSPILKGRVYKILSAEGDNFLLAMSRIIYLDPSLGGVSGMSLGRFHIRQFSDPVSEGLSFWREIWLSSHESWIHALCQEAGNPDLGERTLESFTRLLEDPTTLNIKGGASPTILLKDAIRKALYDEVDKVENSEFREAILLSKTHRDNFILFLKSVEPLFPRFLSELFSSSFLGIPESIIGLIQNSRTIRRQFRKSLSRTLEESFYNSEIHGINRMTQTPQRVGRVWPCSSERADLLREISWGRKVVGTTVPHPSEMLGLLPKSSISCTCGATGGGNPRVSVSVLPSFDQSSFSRGPLKGYLGSSTSMSTQLFHAWEKVTNVHVVKRALSLKESINWFITRNSNLAQTLIRNIMSLTGPDFPLEEAPVFKRTGSALHRFKSARYSEGGYSSVCPNLLSHISVSTDTMSDLTQDGKNYDFMFQPLMLYAQTWTSELVQRDTRLKDSTFHWHLRCNRCVRPIDDITLETSQVFEFPDVSKRISRMVSGAVPHFQKLPDIRLKPGDFESLSGREKSRHIGSAQGLLYSILVAIHDSGYNDGTIFPVNIYGKVSPRDYLRGLARGVLIGSSICFLTRMTNINIYRPLELISGVISYILLRLDNHPSLYIMLREPSLRGEIFSIPQKIPAAYPTTMKEGNRSILCYLQHVLRYEREVITASPENDWLWIFSDFRSSKMTYLTLITYQSHLLLQRVERNLSKSMRANLRQMSSLMRQVLGGHGEDTLESDDDIQRLLKDSLRKTRWVDQEVRHAARTMTGDYSPNKKMSRKAGCSEWVCSAQQVAVSTSANPAPVSELDIRALSKRFQNPLISGLRVVQWATGAHYKLKPILDDLNVFPSLCLVVGDGSGGISRAVLNMFPDAKLVFNSLLEVNDLMASGTHPLPPSAIMSGGDDIVSRVIDFDSIWEKPSDLRNLTTWKYFQSVQKRVNMSYDLIICDAEVTDIASINRITLLMSDFALSIDGPLYLVFKTYGTMLVNPEYRAIQHLSRAFPSVTGFITQMTSSFSSELYLRFSKRGKFFRDAEYLTSSTLREMSLVLFNCSSPKSEMQRARSLNYQDLVRGFPEEIISNPYNEMIITLIDSDVESFLVHKMVDDLELQRGTLSKMAVIIAIMIVFSNRVFNVSKPLTDPLFYPPSDPKILRHFNICCSTMMYLATALGDVPSFARLHDLYNRPITYYFKKQVIRGSIYLSWSWSDDTSVFKRVACNSSLSLSSHWIRLVYKIVKTTRLVGSIEDLSGEVVRHLQGYNRWITLEDIRSRSSLLDYSCL"
实施例4
1.用预定的工作种子批毒种连续制备三批原疫苗。具体步骤如下：
以MOI=0.01 FFU/细胞的接种量将RV CTNCEC25株工作种子批病毒（即CTNCEC25株40代毒种）接种于CEC悬液（按照实施例1所述制备CEC悬液，培养基更换为M2或M3，其细胞数量调整为0.8～1.4×106个细胞/ml）中，混合均匀后分装于细胞瓶或细胞工厂，然后置于33～36℃、5% CO2培养。
待80%以上的细胞出现病变即可收获病毒上清液，收获的病毒上清液经针孔式过滤器（Ф0.60μm，Millipore公司）过滤澄清后，加入β-丙内酯，使其与病毒悬液的最终比例达到1:4000（v/v），充分搅拌混匀后，置于2～8 ℃孵育至少24小时，以确保充分灭活病毒的感染性而不丧失病毒的抗原性。24小时后将已充分灭活的病毒液置于37℃放置2小时，充分水解病毒液中残余的β-丙内酯，即得到待测疫苗，保存于2～8 ℃，以备疫苗效价和血清抗体水平检测。
2.采用NIH法对待测疫苗的效价进行检测，以评估其免疫保护性。
按照狂犬病实验室技术（Laboratory Technologies for Rabies，WHO，1996）和2010年版《中国药典》进行。通过比较保护小鼠免受致死量脑内狂犬病毒侵袭所需的实验疫苗剂量与给予相同保护所需的相应参考疫苗（中国食品药品检定研究院，批号250009-201108，6.6 IU/剂）来确定狂犬病原疫苗的效价。
将参考疫苗用1.0 ml无菌注射用水重溶后，用PBS对其进行5倍梯度稀释，最终取稀释度范围为1:25～1:3125 （即上述4种稀释度，分别指1:25、1:125、1:625、1:3125，下同）进行小鼠免疫。
制备好的待测疫苗也用PBS进行5倍梯度稀释，最终取稀释度范围为1:25～1:3125进行小鼠免疫。
取稀释后的参考疫苗和待测疫苗，通过腹腔途径对小鼠进行初次免疫，每个稀释度至少16只小鼠，每只小鼠0.5 ml疫苗稀释液。初免后7天，按照初次免疫接种的步骤和量对小鼠进行二次免疫。初次免疫后14天，用含5～100 LD50的CVS病毒悬液（来源于中国食品药品检定研究院，初始为CVS-8，经小鼠脑内传2代，即CVS-10）对免疫后的小鼠进行脑内攻击。脑内攻击后逐日观察14天，3天内死亡小鼠记为非特异性死亡。通过Reed-Muench法进行计算效价。发现：
所述待测疫苗和参考疫苗的实际ED50值均在给予实验动物的最高和最低剂量之间。
所述CVS株病毒悬液的攻击量为10 LD50/0.03ml，接受该攻击量病毒悬液的所有实验动物均出现死亡。
测试疫苗的效价按照下列公式进行计算：
测试疫苗相对效力 P=(T/S)×dT/dS×D
其中 上述公式中 P为待测疫苗效价，IU/ml；
T为待测疫苗ED50的倒数；
S为参考疫苗ED50的倒数；
dT为待测疫苗的1次人用剂量，ml；
dS为参考疫苗的1次人用剂量，ml；
D为参考疫苗的效价，IU/ml。
最终结果如表5所示：
表5
批次号 待测疫苗的ED50 参考疫苗的ED50 效价 （IU/ml） CVS攻击量（LD50/0.03ml） 平均效价（IU/ml）
Ⅰ 10-3.02 10-3.03 6.45 10 5.39
Ⅱ 10-2.80 10-2.98 4.36 13
Ⅲ 10-2.94 10-3.03 5.36 10
从上述实验可以得知，待测疫苗的平均效价是5.39 IU/ml，每一检测中应用的CVS株病毒攻击剂量均在5～100 LD50之间。每一次检测中应用的参考疫苗的实际ED50均处于最高和最低稀释度之间。因此可得出结论认为，该疫苗具有免疫保护性，且NIH效价高于WHO和中国现行版药典的质量标准2.5 IU/剂。
3.体内抗体水平的检测
3.1小鼠体内抗体水平的动态变化
按照原疫苗制备工艺制备待测疫苗作为免疫原，通过腹腔途径对12～14 g的SPF级白化昆明小鼠进行初次免疫，每只小鼠0.5ml，初免后7天，按照初次免疫接种的步骤和量对小鼠进行二次免疫，二次免疫后3天、7天、10天和14天分别取5只小鼠，通过眼球动脉采集血样，将采集的血样于37℃孵育1小时使其凝固，然后将凝固后的血样以2000 rpm离心10分钟分离血清，并将血清无菌收集于灭菌试管或冷冻管内。收集后的血清置于56℃灭活30分钟，储存于-70℃备用。
对上述收集的血清进行抗体效价测定，抗体效价测定按照2010年版《中国药典》附录Ⅺ J“狂犬病免疫球蛋白效价测定法”中的 “快速荧光灶抑制试验法”进行。测定结果如表6所示：
表6
取样时间 试验小鼠编号 抗体效价（IU/ml） 平均抗体效价（IU/ml）
3天 小鼠1 9.55 4.886
小鼠2 3.23
小鼠3 1.69
小鼠4 3.44
小鼠5 6.52
7天 *小鼠1 / 6.573
*小鼠2 /
小鼠3 3.86
小鼠4 7.93
小鼠5 7.93
10天 小鼠1 66.95 40.384
小鼠2 14.98
小鼠3 45.89
小鼠4 42.65
小鼠5 31.45
14天 小鼠1 59.3 40.92
小鼠2 15.44
小鼠3 51.44
小鼠4 57.42
小鼠5 21
*表明小鼠血样过于黏稠，离心效果很差。
从表6中可以看出，用本发明毒株制备的原疫苗作为免疫原免疫小鼠，二次免疫后的10天和14天小鼠体内血清平均抗体效价均可达到40 IU/ml以上。
3.2家兔体内抗体水平的检测
按照原疫苗制备工艺制备待测疫苗作为免疫原，通过肌肉途径对1.5～2.0 kg的5只家兔进行初次免疫，每只家兔1.0ml，初免后7天，按照初次免疫接种的步骤和量对家兔进行二次免疫，二次免疫后的14天通过耳部静脉采集血样，将采集的血样于37℃孵育1小时使其凝固，然后将凝固后的血样以2000 rpm离心10分钟分离血清，并将血清无菌收集于灭菌试管或冷冻管内。收集后的血清置于56℃灭活30分钟，储存于-70℃备用。
对上述收集的血清进行抗体效价测定，抗体效价测定按照2010年版《中国药典》附录Ⅺ J“狂犬病免疫球蛋白效价测定法”中的 “快速荧光灶抑制试验法”进行。结果如表7所示：
表7
试验家兔编号 抗体效价（IU/ml） 平均抗体效价（IU/ml）
家兔1 78 41.88
家兔2 24
家兔3 27.5
家兔4 54.3
家兔5 25.6
从表中可以看出，用本发明毒株制备的原疫苗作为免疫原免疫家兔，二次免疫后的14天家兔体内血清平均抗体效价也可达到40 IU/ml以上。
由此可见，如用本申请毒株制备的抗原作为免疫原，二次免疫动物后的10天至14天即可获得较高水平的抗RV血清，可进行抗血清或抗体的制备。

Claims (2)

  1. 一种狂犬病病毒CTN鸡胚细胞适应株，其微生物保藏号为：CGMCC No. 6510。
  2. 权利要求1所述的狂犬病病毒CTN鸡胚细胞适应株在制备狂犬病病毒灭活疫苗中的用途。
PCT/CN2014/075230 2014-04-03 2014-04-14 狂犬病病毒ctn鸡胚细胞适应株 WO2015149386A1 (zh)

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