WO2012159522A1 - 犬流感重组病毒及其制备方法和应用 - Google Patents

犬流感重组病毒及其制备方法和应用 Download PDF

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WO2012159522A1
WO2012159522A1 PCT/CN2012/074722 CN2012074722W WO2012159522A1 WO 2012159522 A1 WO2012159522 A1 WO 2012159522A1 CN 2012074722 W CN2012074722 W CN 2012074722W WO 2012159522 A1 WO2012159522 A1 WO 2012159522A1
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virus
canine influenza
gene
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nucleotide sequence
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PCT/CN2012/074722
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李泽君
滕巧泱
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中国农业科学院上海兽医研究所
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Priority to CA2837126A priority Critical patent/CA2837126A1/en
Priority to KR1020137031403A priority patent/KR20140031912A/ko
Priority to US14/119,366 priority patent/US20140286979A1/en
Priority to JP2014511717A priority patent/JP2014516530A/ja
Priority to EP12790131.2A priority patent/EP2716752A4/en
Publication of WO2012159522A1 publication Critical patent/WO2012159522A1/zh

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Definitions

  • the invention relates to the field of bioengineering technology, in particular to a canine influenza recombinant virus and a preparation method and application thereof.
  • Influenza A is a major infectious disease that threatens human health. Influenza viruses have strict host specificity, and even the spread of the same virus on different hosts is limited by the host community. As early as 2004, the United States first reported that the H3N8 subtype canine influenza virus caused a major outbreak of canine influenza. Through sequence analysis, it was found that the subtype of canine influenza virus was derived from the horse flu virus. Subsequently, the canine flu virus broke out after the horse flu in Australia. In 2008, a dog flu caused by the H3N2 subtype canine influenza virus broke out in South Korea. Through sequence analysis, it was found that the H3N2 subtype of canine influenza virus is avian origin, unlike the Mayuan dog flu virus in Europe and the United States.
  • H3N2 subtype canine influenza virus In 2006-2007, several strains of H3N2 subtype canine influenza virus were isolated from dogs in southern China. Through sequence analysis, these viruses were found to be highly homologous to viruses isolated from Korea. A survey of sera from dogs in southern China found that 6.7% of dog sera were flu-positive. In 2010, the laboratory also isolated a canine influenza virus from East China, named A/canine/Zhejiang/01/2010 (H3N2 subtype, referred to as ZJCIV). Whole genome sequence analysis of the ZJCIV virus revealed that the virus is highly homologous to the South China and South Korea H3N2 canine influenza viruses.
  • ZJCIV virus can infect dogs, and can cause canine disease, which is characterized by decreased appetite, high fever, cough and discharge of purulent secretions in the nasal cavity. After dissection, pulmonary congestion and hemorrhage are found, and the alveoli are filled with inflammatory exudation. Things.
  • the technical problem to be solved by the present invention is to provide a canine influenza recombinant virus comprising the HA and NA genes of the canine influenza virus ZJCIV and six internal genes of the PR8 virus, which can be effectively produced against the H3N2 subtype dog. Vaccine of influenza virus.
  • a canine influenza recombinant virus comprising: HA and NA genes of ZJCIV canine influenza virus, and PA, PB1, PB2, M, NP and NS of PR8 virus Six internal genes,
  • the nucleotide sequence of the canine influenza virus HA gene is selected from the group consisting of:
  • the nucleotide sequence of the canine influenza virus NA gene is selected from the group consisting of:
  • the canine influenza virus HA gene has SEQ ID The nucleotide sequence shown by NO. 3, or the canine influenza virus HA gene has a sequence having 98% or more homology with the nucleotide sequence shown by SEQ ID NO.
  • the canine influenza virus NA gene has SEQ ID The nucleotide sequence shown by NO. 4, or the canine influenza virus NA gene has a sequence having 98% or more homology with the nucleotide sequence shown by SEQ ID NO.
  • the amino acid sequence of ZJCIV canine influenza virus hemagglutinin (HA) activity obtained by deleting, adding, inserting or substituting one or more amino acids in the amino acid sequence shown in NO.
  • HA hemagglutinin
  • An amino acid sequence having ZJCIV canine influenza virus neuraminidase (NA) activity obtained by deleting, adding, inserting or substituting one or more amino acids in the amino acid sequence shown in NO.
  • the HA gene of the ZJCIV canine influenza virus contained in the canine influenza recombinant virus of the present invention is a coding SEQ Nucleotide sequence of the amino acid sequence shown by ID NO.1, or coding with SEQ ID
  • the nucleotide sequence of the amino acid sequence shown in NO. 1 has an amino acid sequence of 98% or more homology
  • the NA gene of the ZJCIV canine influenza virus contained in the canine influenza recombinant virus of the present invention encodes the SEQ ID A nucleotide sequence of the amino acid sequence shown by NO. 2, or a nucleotide sequence encoding an amino acid sequence having 98% or more homology with the amino acid sequence shown by SEQ ID NO.
  • a method for preparing the above-described canine influenza recombinant virus comprising the steps of:
  • the recombinant plasmid of the HA gene and the recombinant plasmid of the NA gene were transfected into 293T cells together with six plasmids containing the internal genes of PR8 virus PA, PB1, PB2, M, NP and NS, respectively, and the transfected cells were cultured;
  • the cultured cell supernatant is inoculated into the chicken embryo, and after culturing for a suitable time in the incubator, the chicken embryo allantoic fluid is harvested, and the hemagglutination property of the allantoic fluid is detected, if there is hemagglutination activity, and the sequence analysis determines that there is no unexpected After the mutation, a canine influenza recombinant virus is obtained.
  • the cultured cell supernatant is inoculated into chicken embryos of 9-11 days old, and the chicken embryo allantoic fluid is harvested after 48-72 hours of incubation in a 37 ° C incubator.
  • the recombinant plasmid uses a PBD vector as an empty vector.
  • an influenza vaccine which is produced by using the above-described canine influenza recombinant virus as a poison.
  • the canine influenza recombinant virus of the invention can produce high virus titer and blood coagulation titer on both chicken embryo and MDCK cells, and can be used as an excellent seed poison for developing a canine influenza vaccine.
  • Example 1 is an RT-PCR electrophoresis pattern of HA and NA of canine influenza virus ZJCIV of Example 1 of the present invention
  • Example 2 is a graph showing the blood coagulation valence of different time after the recombinant virus and ZJCIV vaccinated chicken embryos obtained in Example 3 of the present invention
  • Figure 3 is a graph comparing growth curves of recombinant virus and ZJCIV rescued on chicken embryos in Example 3 of the present invention
  • Example 4 is a graph showing the blood coagulation valence at different times after the recombinant virus rescued in Example 3 of the present invention and ZJCIV inoculated with MDCK cells;
  • Figure 5 is a graph comparing growth curves of recombinant virus and ZJCIV rescued on MDCK cells in Example 3 of the present invention.
  • the A/canine/Zhejiang/01/2010 virus (H3N2 subtype, or ZJCIV) isolated before the laboratory is in the chicken embryo or in the cell. After amplification, the hemagglutination titer was very low.
  • the present invention recombines the main antigenic proteins HA and NA genes of the ZJCIV canine influenza virus with the remaining six internal genes of the PR8 virus, and rescues one strain in the chicken embryo and the cell by the reverse genetic operating system of the influenza virus.
  • a canine influenza recombinant virus that produces high viral titers and hemagglutination titers, which can be used as an excellent virus for the development of a canine influenza vaccine.
  • RNA from canine influenza virus ZJCIV was extracted with Trizol (Invitrogen). Using Reverse Transcription System Kit reverse transcription kit (TakaRa), according to the instructions, using the 12 bp primer 5'-AGCAAAAGCAGG-3' as a specific primer to synthesize the first strand of cDNA. The first strand of the obtained cDNA was used as a template, and sapI-HA-up, sapI-HA-down and sapI-NA-up, and sapI-NA-down were used as primers for upstream and downstream (containing BspQI restriction sites, as shown in Table 1). ), the HA and NA of the fragment ZJCIV were amplified, respectively.
  • the PCR amplification procedure was predenatured at 94 °C for 5 min, entering the following cycle, denaturation at 94 °C for 45 s, annealing at 53 °C for 45 s, extension at 72 °C for 1 min and 45 s, running for 30 cycles, and finally extending at 72 ° C for 10 min.
  • a negative control without template was set.
  • the PCR product was subjected to electrophoresis on a 1.0% agarose gel.
  • the bp HA and the 1400 bp NA match the size of the target fragment.
  • M DNA molecular weight standard
  • 1 ZJCIV HA PCR product
  • 2 ZJCIV NA PCR product.
  • Primer name Primer sequence sapI-HA-up CACACAgctcttctattAGCAAAAGCAGGGG ( SEQ ID NO. 5 ) sapI-HA-down CACACAgctcttcggccAGTAGAAACAAGGGTGTTTTTT ( SEQ ID NO. 6 ) sapI-NA-up CACACAgctcttctattAGCAAAAGCAGGAGT ( SEQ ID NO.7 ) sapI-NA-down CACACAgctcttcggccAGTAGAAACAAGGAGTTTTTT ( SEQ ID NO.8 )
  • the agarose gel of the DNA fragment of interest was excised from the gel under ultraviolet light, and the DNA was recovered using a DNA rapid recovery kit.
  • the preparation tube was placed back in the collection tube and vaccinated at 12000 x g for 1 min. Finally, the preparation tube is placed in a clean 1.5ml. In the EP tube, 30 ul of deionized water was added to the center of the prepared membrane, and the mixture was allowed to stand at room temperature for 1 min, and centrifuged at 12,000 ⁇ g for 1 min to elute the DNA, and stored at -20 ° C until use.
  • the purified PCR product and the PBD vector (preserved in the laboratory) were incubated at 50 ° C for 1 hour under the action of BspQI restriction enzyme (NEB) according to the instructions.
  • the target fragment and the PBD plasmid were digested with a gel recovery kit, and 1 ul of T4 ligase buffer and 1 ul were added.
  • T4 ligase (TakaRa), the reaction system was 10 ul, and mixed. Connect overnight at 16 °C.
  • the ligation and production were transformed into competent cells JM109 (prepared in our laboratory), and plated on Amp-containing LB solid medium under sterile conditions in a sterile condition, and cultured at 37 ° C for 8-20 h.
  • a single colony on LB solid medium was picked and placed in a test tube supplemented with about 3 ml of Amp-containing LB liquid medium, and then fixed on a shaker at 37 ° C for 10 h or overnight.
  • the plasmid extracted by the alkaline extraction method was verified by a PCR method.
  • the plasmids identified as positive were sequenced and aligned using DNAstar sequence analysis software.
  • the results showed that the recombinant plasmids PBD-ZJCIVHA and PBD-ZJCIVNA were successfully constructed, and the HA gene sequence was as SEQ. As shown in ID No. 3, the NA gene sequence is shown in SEQ ID NO.
  • the plasmid was extracted with an ultra-pure plasmid extraction kit (OMEGA).
  • OEGA ultra-pure plasmid extraction kit
  • the procedure was as follows: 1) Preservation of glycerol bacteria by inoculating loops (containing plasmids PBD-ZJCIVHA, PBD-ZJCIVNA, PBD-PR8M, PBD-PR8PB1, PBD- PR8PB2, PBD-PR8PA, PBD-PR8NS, PBD-PR8NP, the last six plasmids are stored in the laboratory), containing Amp
  • the surface of the LB plate was streaked and allowed to stand overnight at 37 °C.
  • the above ultra-purified plasmids including PBD-ZJCIVHA, PBD-ZJCIVNA, PBD-PR8M, PBD-PR8PB1, PBD-PR8PB2, PBD-PR8PA, PBD-PR8NS and PBD-PR8NP, were passed through a suitable amount of liposome 2000. Transfected into 3.5 cm diameter 293T cells. 6h after transfection, the cell supernatant was discarded and 2ml was added. The culture medium of OPTI-MEM (Invitrogen) was cultured in a CO2 incubator at 37 ° C for 72 h.
  • OPTI-MEM Invitrogen
  • RNA of the allantoic fluid of the recombinant virus was extracted with Trizol and reverse transcribed with a 12 bp primer to obtain the first strand of cDNA.
  • cDNA first strand as template, sapI-HA-up, sapI-HA-down and sapI-NA-up, sapI-NA-down as upstream and downstream primers, PCR amplification of ZJCIV HA and NA fragments were identified by electrophoresis on 1% agarose.
  • ZJCIV HA and ZJCIV NA The PCR product was sent to the company for sequencing. RESULTS: Two segments appeared on the agarose gel, which were approximately 1700 bp and 1400, respectively. Bp, exactly the same size as the destination. The sequencing results further confirmed that the PCR product was indeed a ZJCIV HA and NA fragment.
  • the virus-containing chicken embryo allantoic fluid was diluted 10 times, and 10-6 to 10-10 dilutions were inoculated into 5 9-11 day old SPF chicken embryos, and incubation was continued for 48 hours at 37 °C.
  • the blood coagulation activity of the infected embryonic allantoic fluid was measured to determine whether it was infected, and the EID50 (the half infection amount of the chicken embryo) was calculated by the Reed-Muench method. Results: The EID50 of the rescued recombinant virus and ZJCIV was 107.5/100ul, respectively. And 106.5/100ul.
  • the virus-adsorbed cells were further cultured in a CO2 incubator for 72 hours, and then the hemagglutination activity of each well was measured, and TCID50 (half the amount of tissue cells infected) was calculated by the Reed-Muench method. Results: The TCID50 of the rescued recombinant virus and ZJCIV was 106.5/100ul, respectively. And 105.5/100ul.
  • 6h, 12h, 24h, 36h, 48h, and 72h after inoculation three inoculated SPF chicken embryos were taken out, and their allantoic fluid was collected and their blood coagulation titers were determined (Fig. 2).
  • the allantoic fluid collected at each time period was diluted 10 times, and each of the diluted virus liquids was inoculated with 3 9-11 day old SPF chicken embryos, and the amount of poison was 100 ul/piece.
  • the hemagglutination activity of the chicken embryo allantoic fluid was measured 48 h after the poisoning.
  • the virus content of the allantoic fluid was collected at different times, and the growth curve of the virus was drawn (Fig. 3).
  • the rescued recombinant virus and ZJCIV were compared on the chicken embryo. growing situation. The results are shown in Figure 2.
  • the allantoic fluid of ZJCIV and recombinant virus was not hemagglutinating.
  • the hemagglutination titer of the recombinant virus reached 211, which was significantly higher than the blood coagulation titer of ZJCIV.
  • ZJCIV and the rescued recombinant virus failed to detect the virus when they were exposed for 6 hours.
  • the titer of the rescued recombinant virus was significantly higher than that of ZJCIV, and reached the highest peak after 48 hours of exposure.
  • Recovered recombinant virus and ZJCIV Dilute to 100 TCID50 and inoculate 3 bottles of MDCK cells to 80% T25 cell vials according to this dilution.
  • the cell supernatants were collected and their blood coagulation titers were determined (Fig. 4).
  • the virus content in the supernatant of the cells collected at each time was titrated as follows: The collected cell supernatant was diluted 10-fold, and each diluted virus solution was inoculated into 3 wells to grow up to 80% of MDCK cells on a 24-well cell plate.
  • the blood coagulation activity was measured 48 h after the poisoning, and the virus content (TCID50) in the cell supernatant collected at each time was calculated, and the growth curve of the virus was plotted according to the TCID50 (Fig. 5), and the rescued recombinant virus and ZJCIV were compared in MDCK. Growth on cells.
  • the results of blood coagulation titer are shown in Figure 4. Within 12 hours after exposure, the cell supernatant of ZJCIV has no hemagglutination activity, while the cell supernatant of the recombinant virus has hemagglutination activity and peaks at 36 h after exposure. It tends to be relatively stable.
  • the hemagglutination activity of the recombinant virus was significantly higher than that of ZJCIV throughout the infection.
  • the results of virus titer are shown in Figure 5.
  • ZJCIV and the rescued recombinant virus failed to detect the virus when exposed to poison for 6 hours.
  • the rescued recombinant virus could be detected 12 hours after the poisoning, and ZJCIV was not detected until the virus was received. It can only be detected after 24 hours.
  • the virus titer of the recombinant virus and ZJCIV peaked and then began to decline.
  • the viral titer of the recombinant virus was significantly higher than the viral titer of ZJCIV throughout the cell infection process.

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Abstract

本发明公开一种犬流感重组病毒，该重组病毒包含：ZJCIV犬流感病毒的HA和NA基因、以及PR8病毒的PA、PB1、PB2、M、NP和NS六个内部基因。本发明还公开了犬流感重组病毒的制备方法及应用。本发明的犬流感重组病毒，在鸡胚和MDCK细胞上均能产生很高的病毒滴度和血凝滴度，可作为研制犬流感疫苗的优良种毒。

Description

犬流感重组病毒及其制备方法和应用
技术领域
本发明涉及生物工程技术领域，尤其涉及一种犬流感重组病毒及其制备方法和应用。
背景技术
A型流感病毒是一类威胁人类健康的重要传染病。流感病毒具有严格的宿主特异性，即便是相同病毒在不同的宿主上的传播也受到宿主界限制。早在2004年，美国首次报道H3N8亚型犬流感病毒引起犬流感的大爆发，通过序列分析，发现该亚型的犬流感病毒是从马流感病毒衍变而来的。随后，澳大利亚发生马流感后也爆发犬流感病毒。2008年，韩国爆发了由H3N2亚型犬流感病毒引起的犬流感，通过序列分析，发现H3N2亚型的犬流感病毒是禽源的，不同于欧美国家的马源犬流感病毒。
2006-2007年间，中国华南地区发病犬体内分离获得数株H3N2亚型犬流感病毒，通过序列分析，发现这些病毒与韩国分离到病毒高度同源。对华南地区宠物狗的血清进行调查发现，6.7%的狗血清呈流感阳性。2010年，本实验室从华东地区也分离获得一株犬流感病毒，命名为A/canine/Zhejiang/01/2010(H3N2亚型，简称ZJCIV)。对ZJCIV病毒的全基因组序列分析发现，该病毒与华南及韩国H3N2犬流感病毒高度同源。动物感染实验发现ZJCIV病毒可以感染狗，并可以引起犬发病，表现为食欲下降、高热、咳嗽和鼻腔中排出脓性分泌物等症状，解剖后发现肺淤血、出血，肺泡中充满炎性渗出物。
2009年6月，Intervet公司研究成功犬流感病毒灭活苗，在美国已经上市，但是中国流行的犬流感病毒与之前美国流行的犬流感病毒抗原性有差别，二者起源于不同的H3流感病毒分支。因而，研制针对H3N2亚型犬流感病毒流行株的疫苗，对于犬流感的预防控制具有重要的实际意义。
发明内容
本发明要解决的技术问题是提供一种犬流感重组病毒，其包含犬流感病毒ZJCIV的HA和NA基因、以及PR8病毒的6个内部基因，该重组病毒能制成有效的针对H3N2亚型犬流感病毒的疫苗。
此外，还需要提供一种上述犬流感重组病毒的制备方法和应用。
为了解决上述技术问题，本发明通过如下技术方案实现:
在本发明的一个方面，提供了一种犬流感重组病毒，该重组病毒包含：ZJCIV犬流感病毒的HA和NA基因、以及PR8病毒的PA、PB1、PB2、M、NP和NS 六个内部基因，
所述犬流感病毒HA基因的核苷酸序列选自：
(1)编码SEQ ID NO.1所示氨基酸序列的核苷酸序列；
(2)编码与SEQ ID NO.1所示氨基酸序列具有98%以上同源性的氨基酸序列的核苷酸序列；
所述犬流感病毒NA基因的核苷酸序列选自：
(1)编码SEQ ID NO.2所示氨基酸序列的核苷酸序列；
(2)编码与SEQ ID NO.2所示氨基酸序列具有98%以上同源性的氨基酸序列的核苷酸序列。
优选的，所述犬流感病毒HA基因具有SEQ ID NO.3所示的核苷酸序列，或者所述犬流感病毒HA基因具有与SEQ ID NO.3所示核苷酸序列具有98%以上同源性的序列。
优选的，所述犬流感病毒NA基因具有SEQ ID NO.4所示的核苷酸序列，或者所述犬流感病毒NA基因具有与SEQ ID NO.4所示核苷酸序列具有98%以上同源性的序列。
在本发明中，与SEQ ID NO.1所示氨基酸序列具有98%以上同源性的氨基酸序列，包括SEQ ID NO.1所示氨基酸序列中缺失、添加、插入或取代一个或多个氨基酸所得到的具有ZJCIV犬流感病毒血凝素（HA）活性的氨基酸序列。
在本发明中，与SEQ ID NO.2所示氨基酸序列具有98%以上同源性的氨基酸序列，包括SEQ ID NO.2所示氨基酸序列中缺失、添加、插入或取代一个或多个氨基酸所得到的具有ZJCIV犬流感病毒神经氨酸酶（NA）活性的氨基酸序列。
HA和NA是流感病毒的两个重要表面抗原，流感病毒的抗原变异主要就是指HA和NA发生变异，尤其HA变得更快。因此，本发明犬流感重组病毒中包含的ZJCIV犬流感病毒的HA基因为编码SEQ ID NO.1所示氨基酸序列的核苷酸序列，或编码与SEQ ID NO.1所示氨基酸序列具有98%以上同源性的氨基酸序列的核苷酸序列；本发明犬流感重组病毒中包含的ZJCIV犬流感病毒的NA基因为编码SEQ ID NO.2所示氨基酸序列的核苷酸序列，或编码与SEQ ID NO.2所示氨基酸序列具有98%以上同源性的氨基酸序列的核苷酸序列。
在本发明的另一方面，还提供了一种上述犬流感重组病毒的制备方法，包括以下步骤：
构建分别包含ZJCIV犬流感病毒HA基因和NA基因的重组质粒；
将所述HA基因的重组质粒和NA基因的重组质粒，与分别包含PR8病毒PA、PB1、PB2、M、NP、NS内部基因的六个质粒一起转染293T细胞，培养转染后的细胞；
将培养的细胞上清接种于鸡胚，在孵化器内培养合适时间后，收获鸡胚尿囊液，检测该尿囊液的血凝性，如果有血凝活性，并且经过序列分析确定没有非预期突变后，即获得犬流感重组病毒。
优选的，将培养的细胞上清接种于9-11日龄鸡胚，在37℃孵化器内培养48-72小时后，收获鸡胚尿囊液。
所述重组质粒采用PBD载体作为空载体。
在本发明的另一方面，还提供了一种上述犬流感重组病毒在制备预防或治疗犬流感的病毒灭活疫苗中的应用。
在本发明的另一方面，还提供了一种流感疫苗，该疫苗以上述犬流感重组病毒为种毒而制成。
本发明犬流感重组病毒，在鸡胚和MDCK细胞上均能产生很高的病毒滴度和血凝滴度，可作为研制犬流感疫苗的优良种毒。
附图说明
下面结合附图和具体实施方式对本发明作进一步详细的说明。
图1是本发明实施例1犬流感病毒ZJCIV的HA和NA的RT-PCR电泳图；
图2是本发明实施例3救获的重组病毒和ZJCIV接种鸡胚后不同时间的血凝效价图；
图3是本发明实施例3救获的重组病毒和ZJCIV在鸡胚上生长曲线比较图；
图4是本发明实施例3救获的重组病毒和ZJCIV接种MDCK细胞后不同时间的血凝效价图；
图5是本发明实施例3救获的重组病毒和ZJCIV在MDCK细胞上生长曲线比较图。
具体实施方式
下列实施例中，未注明具体条件的实验方法，通常按常规条件，如《精编分子生物学实验指南》(F. M. 奥斯伯, R. E. 金斯顿, J. G. 塞德曼等主编，马学军, 舒跃龙的译. 北京：科学出版社，2004)中所述的方法进行。
犬流感病毒疫苗研制的重要前提之一是有优良的种毒，本实验室之前分离到的A/canine/Zhejiang/01/2010病毒(H3N2亚型，简称ZJCIV)无论在鸡胚上还是在细胞上扩增后，血凝滴度均非常低。为此，本发明将ZJCIV犬流感病毒主要的抗原蛋白HA、NA基因与PR8病毒其余六个内部基因重组在一起，通过流感病毒的反向遗传操作系统救获一株在鸡胚和细胞均能产生很高的病毒滴度和血凝滴度的犬流感重组病毒，该重组病毒可作为研制犬流感疫苗的优良种毒。
实施例1 重组质粒的构建与鉴定
1、PCR扩增
用Trizol (Invitrogen)抽提犬流感病毒ZJCIV的总RNA。采用Reverse Transcription System kit反转录试剂盒(TakaRa)，根据其说明书，用12bp引物5'-AGCAAAAGCAGG-3'为特异性引物，合成cDNA第一链。以获得的cDNA的第一链为模板，用sapI-HA-up、sapI-HA-down和sapI-NA-up、sapI-NA-down为上下游引物(含有BspQI酶切位点，如表1)，分别扩增出片段ZJCIV的HA和NA。PCR扩增程序为94℃预变性5min，进入以下循环，94℃变性45s，53℃退火45s，72℃延伸1min45s，运行30个循环，最后再72℃延伸10min。同时设无模板的阴性对照。反应结束后，PCR产物在1.0%琼脂糖凝胶上进行电泳实验。结果所图1所示，有两条PCR的条带出现，大小分别约为1700 bp的HA和1400 bp的NA，目的片段大小相符。在图1中，M: DNA分子量标准；1: ZJCIV HA PCR产物；2: ZJCIV NA PCR产物。
表1 A型流感病毒 HA和NA基因的通用引物
引物名称 引物序列
sapI-HA-up CACACAgctcttctattAGCAAAAGCAGGGG （ SEQ ID NO.5 ）
sapI-HA-down CACACAgctcttcggccAGTAGAAACAAGGGTGTTTT （ SEQ ID NO.6 ）
sapI-NA-up CACACAgctcttctattAGCAAAAGCAGGAGT （ SEQ ID NO.7 ）
sapI-NA-down CACACAgctcttcggccAGTAGAAACAAGGAGTTTTTT （ SEQ ID NO.8 ）
2、PCR产物的割胶回收
电泳结束后在紫外光下从凝胶上切下目的DNA片段的琼脂糖凝胶，用DNA快速回收试剂盒回收DNA。具体方法如下：在紫外灯下切下含有目的DNA的琼脂糖凝胶，用纸巾吸尽凝胶表面液体并切碎，放入一无菌的1.5ml的EP管中，加入3倍凝胶体积（100mg=100ul体积）的Buffer DE-A（凝胶液），混合均匀后于75℃加热，间断混合（2-3min），直至凝胶块完全熔化（约6-8min）。加入0.5个Buffer DE-A体积的Buffer DE-B（结合液），混合均匀；当回收的DNA片段小于400bp时，加入1个凝胶体积的异丙醇。将混合液转移到DNA制备管中，12000×g 离心1min，倒掉收集管中的废液。将制备管置回收集管中，加入500ul Buffer W1（洗涤液），12000×g 离心30s，倒掉收集管中的废液。将制备管置回收集管中，加入700ul Buffer W2（去盐液），12000×g 离心1min，倒掉收集管中的废液，以同样的方法再洗一次。将制备管置回收集管中，12000×g 空离1min。最后将制备管置于洁净的1.5ml EP管中，在制备膜中央加入30ul的去离子水，室温静置1min，12000×g 离心1min洗脱DNA，置于-20℃保存备用。
3、酶切、连接及转化
纯化的PCR产物和PBD载体（本实验室保存）在BspQI限制性内切酶（NEB）的作用下，按照说明书，在50℃温育1个小时。用胶回收试剂盒回收目的片段与PBD质粒的酶切产物，加入1ul的T4连接酶的缓冲液和1ul T4连接酶(TakaRa)，连接的反应体系为10ul，混匀。16℃过夜连接。连接产化转化于感受态细胞JM109（本实验室制备），并在无菌条件下超净台中涂布于含Amp的LB固体培养基上，37℃培养8-20h。
4、重组质粒的鉴定
挑取LB固体培养基上的单个菌落，放入加有约3ml含Amp的LB液体培养基的试管中，然后固定在摇床上37℃振荡培养10h或过夜。将菌液用碱性抽提法抽提的质粒，用PCR方法进行验证。鉴定为阳性的质粒进行序列测定，用DNAstar序列分析软件比对。结果发现，重组质粒PBD-ZJCIVHA和PBD-ZJCIVNA构建成功，HA基因序列如SEQ ID NO. 3所示，NA基因序列如SEQ ID NO.4所示。
实施例2 重组PR8病毒的拯救
1、转染质粒准备
用超纯质粒抽提试剂盒（OMEGA）抽提质粒，操作步骤如下：1）用接种环蘸取保存甘油菌(含质粒PBD-ZJCIVHA、PBD-ZJCIVNA、PBD-PR8M、PBD-PR8PB1、PBD-PR8PB2、PBD-PR8PA、PBD-PR8NS、PBD-PR8NP，后六个质粒为实验室保存)，在含Amp LB平板表面划线，37 ℃静置过夜；2）挑取单菌落，接种到5ml Amp LB培养基中，37 ℃振荡培养至OD600值为1.0-1.5； 3）收集3ml过夜培养物，离心彻底去除培养基；4）悬浮菌体：用0.25ml含RNase A 的溶液SolutionI悬浮菌体，直至成匀浆状；5）裂解细菌：再加入0.25ml 溶液SolutionII，颠倒5次以轻轻混匀；6）中和：加入0.125ml 溶液Buffer N3，立刻颠倒5次以轻轻混匀,直至形成白色絮状沉淀，室温12,000×g，离心10min；7）小心将上清倒入干净的1.5ml离心管中，加入0.1倍体积的ETR Solution(蓝色)至上清液中，颠倒7-10次，然后冰浴10min；8)加入42℃水浴5min，再次混浊，室温12,000×g，离心3 min，ETR溶液将在离心管底部形成一个蓝色分层；9)上清移入到新的1.5ml离心管中，加0.5倍体积的无水乙醇，颠倒6-7次，室温放置1-2min；10)上述混合液倒入一个事先用2ml 溶液E4平衡好的HiBand DNAMini柱，放在一个2ml的收集管上，室温10,000×g，离心1min，使裂解液流过柱子；11)弃去收集管中的液体，将剩余的混合液加入到柱子中，室温10,000×g，离心1min，使裂解液完全通过柱子；加入500ul Buffer HB到柱子上，室温下10,000离心1min，洗涤柱子，确保除去残余的蛋白质，以得到高质量的DNA；12)弃液体，用700ulDNA Wash buffer洗涤柱子，室温下10,000×g，离心1min，弃去液体；13)重复，再加DNA Wash buffer；14)弃液体，空转，室温12,000×g离心2min，去液体；15)把柱子置于一个干净的1.5ml离心管中，加入30-50ul的无内毒素洗脱液到柱子上，室温静置2min，室温12,000×g离心1min以洗脱DNA，可洗两次；16）电泳检测，并用Nanodrop 2000c 紫外可见光分光光度计测量OD260、OD280，估算DNA含量与纯度。结果：获得了足量的转染所需质粒。
2、转染293T细胞
将上述超纯度抽提的质粒，包括PBD-ZJCIVHA、PBD-ZJCIVNA、PBD-PR8M、PBD-PR8PB1、PBD-PR8PB2、PBD-PR8PA、PBD-PR8NS和PBD-PR8NP，通过适量的脂质体2000共转染于3.5cm直径的293T细胞中。转染后6h，弃去细胞上清，加入2ml OPTI-MEM (Invitrogen)培养液，并放于37℃的CO2培养箱中培养72h。
3、拯救获得重组的PR8病毒
转染48h后的细胞上清，接种于9-11日龄SPF鸡胚(北京梅里亚维通实验动物技术有限公司)，用石蜡封口后置于37℃孵化器内培养。48-72h放入4℃过夜，取出，收获鸡胚尿囊液。尿囊液用血凝试验测定有无凝集活性。结果：尿囊液能使红细胞凝集，表明已成功拯救出一株含ZJCIV HA和NA基因的PR8重组病毒。
4、重组病毒的鉴定
用Trizol抽提重组病毒的尿囊液总RNA，并用12bp引物反转录，获得cDNA第一链。以cDNA第一链为模板，用sapI-HA-up、sapI-HA-down和sapI-NA-up、sapI-NA-down为上下游引物，PCR扩增ZJCIV HA和NA片段，并用1%琼脂糖电泳鉴定。此外，将ZJCIV HA和ZJCIV NA PCR产物送公司测序。结果：在琼脂糖电泳胶上出现两条条段，大小分别约为1700 bp 和1400 bp，与目的大小完全一致。测序结果也进一步证实PCR产物确实为ZJCIV HA和NA片段。
实施例3 救获的重组病毒生长特性鉴定
1、救获的重组病毒和ZJCIV的EID50测定
含病毒的鸡胚尿囊液按照10倍倍比稀释，将10-6～10-10各稀释度分别接种到5枚9-11日龄的SPF鸡胚中，37℃继续孵化48h。通过测定感染胚尿囊液的血凝活性来判断其是否感染，利用Reed-Muench法计算EID50（鸡胚半数感染量）。结果：救获的重组病毒和ZJCIV的EID50分别为107.5/100ul 和106.5/100ul。
2、救获的重组病毒和ZJCIV的TCID50测定
从1：10-3开始10倍稀释，把不同稀释度的重组病毒和ZJCIV接毒于长满单层MDCK细胞的48孔板中，接毒的过程：先用PBS清洗MDCK细胞两遍，然后在每一孔加入100ul病毒，每个稀释度做3个重复，把48孔板放入37℃ CO2培养箱让病毒吸附到细胞上，其中每过20min左右摇晃一次细胞，1.5h-2h后把细胞培养板中的病毒弃掉，用PBS清洗细胞两次，然后加入无血清培养基300ul。吸附了病毒的细胞在CO2培养箱继续培养72h，然后测定每一个孔的血凝活性，利用Reed-Muench法计算TCID50（组织细胞半数感染量）。结果：救获的重组病毒和ZJCIV的TCID50分别为106.5/100ul 和105.5/100ul。
3、救获的重组病毒和ZJCIV在鸡胚上生长特性比较
救获的重组病毒和ZJCIV 稀释为100EID50，按照这个稀释度分别接种18枚9-11日龄的SPF鸡胚。在接毒后6h，12h，24h，36h，48h，72h时，分别取出3枚接种过的SPF鸡胚，收集其尿囊液并测定它们的血凝效价(图2)。把收集的各个时间段的尿囊液，按10倍稀释，每个稀释度的病毒液接种3枚9-11日龄的SPF鸡胚，接毒量为100ul/枚。接毒后48h测定鸡胚尿囊液的血凝活性，计算不同时间收集尿囊液的病毒含量，并绘制病毒的生长曲线（图3），比较救获的重组病毒和ZJCIV在鸡胚上的生长情况。结果如图2所示，接毒后12h内，ZJCIV和重组病毒的尿囊液均无血凝性。接毒后48h，重组病毒的血凝效价达到211，明显高于ZJCIV的血凝效价。如图3所示结果，ZJCIV和救获的重组病毒在接毒6h时，均未能检测到病毒。接毒12h后，救获的重组病毒病毒滴度明显高于ZJCIV，并于接毒48h后达到最高峰。
4、救获的重组病毒和ZJCIV在MDCK细胞上生长特性的比较
救获的重组病毒和ZJCIV 稀释为100TCID50，按照这个稀释度各接种3瓶MDCK细胞长至80%的T25细胞瓶。在接毒后6h，12h，24h，36h，48h，72h时，分别收集其细胞上清并测定它们的血凝效价(图4)。滴定各个时间收集的细胞上清中的病毒含量，具体如下：把收集的细胞上清按10倍稀释，每个稀释度的病毒液接种3孔在24孔细胞板上长至80%的MDCK细胞，接毒后48h测定血凝活性，计算各个时间收集的细胞上清中的病毒含量（TCID50），根据此TCID50绘出病毒的生长曲线（图5），比较救获的重组病毒和ZJCIV在MDCK细胞上的生长情况。血凝效价的结果如图4所示，接毒后12h内，ZJCIV的细胞上清无血凝活性，而重组病毒的细胞上清有血凝活性，并在接毒后36h达到高峰，随后趋于相对稳定。在整个感染过程中，重组病毒血凝活性明显高于ZJCIV的血凝活性。病毒滴度的结果如图5所示，ZJCIV和救获的重组病毒在接毒6h时，未能检测到病毒，接毒后12h，救获的重组病毒就可以检测到，而ZJCIV直到接毒后24h才能检测到。接毒后36h，重组病毒和ZJCIV的病毒滴度达到高峰，随后便开始下降。在整个细胞感染过程中，重组病毒的病毒滴度明显高于ZJCIV的病毒滴度。
以上所述实施例仅表达了本发明的实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (8)

  1. 1.一种犬流感重组病毒，其特征在于，该重组病毒包含：ZJCIV犬流感病毒的HA和NA基因、以及PR8病毒的PA、PB1、PB2、M、NP和NS 六个内部基因，
    所述犬流感病毒HA基因的核苷酸序列选自：
    (1)编码SEQ ID NO.1所示氨基酸序列的核苷酸序列；
    (2)编码与SEQ ID NO.1所示氨基酸序列具有98%以上同源性的氨基酸序列的核苷酸序列；
    所述犬流感病毒NA基因的核苷酸序列选自：
    (1)编码SEQ ID NO.2所示氨基酸序列的核苷酸序列；
    (2)编码与SEQ ID NO.2所示氨基酸序列具有98%以上同源性的氨基酸序列的核苷酸序列。
  2. 2.根据权利要求1所述的犬流感重组病毒，其特征在于，所述犬流感病毒HA基因具有SEQ ID NO.3所示的核苷酸序列，或者所述犬流感病毒HA基因具有与SEQ ID NO.3所示核苷酸序列具有98%以上同源性的序列。
  3. 3.根据权利要求1所述的犬流感重组病毒，其特征在于，所述犬流感病毒NA基因具有SEQ ID NO.4所示的核苷酸序列，或者所述犬流感病毒NA基因具有与SEQ ID NO.4所示核苷酸序列具有98%以上同源性的序列。
  4. 4.一种权利要求1所述犬流感重组病毒的制备方法，其特征在于，包括以下步骤：
    构建分别包含ZJCIV犬流感病毒HA基因和NA基因的重组质粒；
    将所述HA基因的重组质粒和NA基因的重组质粒，与分别包含PR8病毒PA、PB1、PB2、M、NP、NS内部基因的六个质粒一起转染293T细胞，培养转染后的细胞；
    将培养的细胞上清接种于鸡胚，在孵化器内培养合适时间后，收获鸡胚尿囊液，检测该尿囊液的血凝性，如果有血凝活性，并且经过序列分析确定没有非预期突变后，即获得犬流感重组病毒。
  5. 5.根据权利要求4所述的制备方法，其特征在于，所述重组质粒采用PBD载体作为空载体。
  6. 6.根据权利要求4所述的制备方法，其特征在于，将培养的细胞上清接种于9-11日龄鸡胚，在37℃孵化器内培养48-72小时后，收获鸡胚尿囊液。
  7. 7.权利要求1所述犬流感重组病毒在制备预防或治疗犬流感的病毒灭活疫苗中的应用。
  8. 8.一种流感疫苗，其特征在于，以权利要求1所述犬流感重组病毒为种毒而制成。
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