AIV地方分离株（H9N2）NP和M1基因的克隆及表达载体的构建
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摘要
禽流感（Avian Influenza, AI）是由正粘病毒科A型流感病毒引起的禽类感染和/或疾病综合征，该病首次于1878年在意大利报道，至今已遍布全世界。鸡、火鸡、鸭和鹌鹑等家禽及野鸟、水禽、海鸟等均可感染禽流感。禽流感病毒亚型众多，极易发生基因突变、重组和重排，具有高度的变异性，给禽流感的免疫和诊断带来了极大的困难。自1994年我国内地广东省首次分离到H9N2 AIV以来，H9N2 AIV在我国已广泛存在，制约了养禽业的发展。由于AIV的NP和M1结构高度保守，具有型特异性，均可作为AIV分类和诊断的基础，因此，从H9N2 AIV M1和NP基因作为切入点来研究禽流感的免疫和诊断不失为一个好的方法和途径。
本实验用接种鸡胚的病毒增殖方法大量增殖禽流感地方分离株A/Chicken/Mudan jiang/0823/00(H9N2)（缩写为MJ00）。增殖病毒经差速离心进行纯化，经超速离心浓缩。根据AIV基因组特点设计1条通用引物T-1，反转录获取cDNA。根据Genbank发表的AIV NP、M1基因核苷酸序列，设计两对特异性引物NP-u/NP-l、M1-u/M1-l用以扩增核蛋白NP、基质蛋白M1基因。通过PCR技术，从病毒基因反转录产物cDNA中扩增出核蛋白NP、基质蛋白M1病毒完整基因片段，与pMD18-T克隆载体连接，构建重组质粒pMD18-T-NP、pMD18-T-M1。提取重组质粒经PCR鉴定和酶切鉴定后，对插入片段进行序列测定及分析。结果表明，禽流感病毒分离株NP基因全长为1497bp，包含完整的ORF，编码498个氨基酸。M1基因 全长为759bp，包含完整的ORF，编码252个氨基酸。NP和M1基因测序结果已提交到Genbank,检索号为AY496851(NP)、AY496852 (M1)。与国内已发表的H9N2亚型禽分离株的NP和M1基因核苷酸序列和氨基酸同源性进行比较，结果表明，NP核苷酸同源性在90.51%-99.73%之间，推导氨基酸同源性在95.38%-99.40%之间；M1核苷酸同源性在90.78%-99.08%之间，推导氨基酸同源性在95.63%-100%之间，表明A型AIV的NP基因及M1基因的高度保守性。分离株MJ00与A/Chicken/GuangDong/11/97(H9N2)同源性最高，亲缘关系最近。其中，NP基因核苷酸同源性为99.73%，仅有4个核苷酸发生变异，氨基酸同源性为99.40%，仅有3个氨基酸发生变异。M1基因核苷酸同源性为99.08%，仅有7个核苷酸发生变异，氨基酸同源性为100%。系统进化树分析表明，分离株NP和M1基因均属于H9N2 AIV欧亚分支的Y280-like亚分支。
将NP基因和M1基因分别插入pGEX-6p-1和PET-30a原核表达载体当中，构建了分别含有NP、M1基因的原核表达载体pGEX-6p-NP、PET-30a-NP、PET-30a-M1，为NP、M1基因的表达及进一步研究AIV NP与宿主特异性的关系、M1基因在病毒感染中的作用及禽流感的诊断和免疫奠定了一定的分子生物学基础。
Avian influenza (AI) was an infection and /or disease syndrome caused by avian influenza A virus (AIV), which was firstly reported in 1878 in Italy. AI had spread all over the world. Poultry and wild fowl both were infected by AIV. AIV had numerous subtypes and its genome had the characteristics of recombinant and high degree of variation, which took difficulty to the prevention and diagnosis of AI. In China, the epidemic AI among poultry was reported in 1994 in Guangdong, AIV H9N2 subtype had been the main virus type, and which had made more danger to chicken. The nucleoprotein (NP) and matrix protein M1 (M1) of AIV were of type specificity and had high conservative sequence, both of which could be the foundation of classification and diagnosis. So we study NP and M1 from molecular level so as to prevent and diagnose AI.
In this research, the AIV（H9N2） isolate was propagated in chicken embryos. The virus was purified by ultracentrifuge. According to the characteristics of AIV genome and the NP gene and M1 gene sequence published by Genbank, a primer T-1 was designed to amplify the cDNA by reverse transcript. Other primers NP-u/NP-1 and M1-u/M1-1 were designed to amplify the NP gene and M1 gene. The PCR product was cloned into pMD18-T vector. The positive recombinant clone was identified by PCR and endonuclease digest. Then the inserting DNA fragment was sequenced and analysed. The NP gene of AIV (H9N2） isolate was 1497bp, which encodes 498 amino acids, M1 gene was 759bp in length, encoding 252 amino acids. The sequences of NP gene and M1 gene had been submitted to Genbank, the number were AY496851(NP) and AY496852(M1). Compared with other published NP and M1 cDNA sequences, it showed that the homology of NP gene was from 90.51% to 99.73% and the homology of amino acids was from 95.38% to 99.40%, the homology of M1 gene was from 90.78% to 99.08% and the homology of amino acids was from 95.63% to 100%. The result showed that the NP gene and M1 gene had high conservative sequence. The result also showed Avian Influenza Virus A/chicken/Mudanjiang/0823/00(H9N2) strain had the close relationship with A/Chicken/Guangdong/11/97(H9N2). The homology of NP gene was 99.73% and the homology of amino acids was 99.40%, only 3 amino acids was different, the homology of M1 gene was from 90.78% to 99.08% and the homology of amino acids was from 95.63% to 100%. The analysis of phylogenetic tree showed that NP and M1 gene of the isolate were Y280-like sublineage of Eurasian avian lineage.
The NP and M1 Gene were cloned into the prokaryotic expression vector pGEX-6p-1 and PET-30a respectively. The recombinant vector was identified by endonuclease. The result showed that the target gene was subcloned successfully into the expression vector and construct the recombinant expression vector pGEX-6p-NP, PET-30a-NP and PET-30a- M1.The expression vectors containing NP and M1 gene facilitates the study for the relationship both NP and host particularity, the function of M1 in virus infection and prevention and diagnosis of AI.
Candidate: Guan Xueting
Major: Preventive Veterinary Medicine
Supervisor: Prof. Wang Junwei
本实验用接种鸡胚的病毒增殖方法大量增殖禽流感地方分离株A/Chicken/Mudan jiang/0823/00(H9N2)（缩写为MJ00）。增殖病毒经差速离心进行纯化，经超速离心浓缩。根据AIV基因组特点设计1条通用引物T-1，反转录获取cDNA。根据Genbank发表的AIV NP、M1基因核苷酸序列，设计两对特异性引物NP-u/NP-l、M1-u/M1-l用以扩增核蛋白NP、基质蛋白M1基因。通过PCR技术，从病毒基因反转录产物cDNA中扩增出核蛋白NP、基质蛋白M1病毒完整基因片段，与pMD18-T克隆载体连接，构建重组质粒pMD18-T-NP、pMD18-T-M1。提取重组质粒经PCR鉴定和酶切鉴定后，对插入片段进行序列测定及分析。结果表明，禽流感病毒分离株NP基因全长为1497bp，包含完整的ORF，编码498个氨基酸。M1基因 全长为759bp，包含完整的ORF，编码252个氨基酸。NP和M1基因测序结果已提交到Genbank,检索号为AY496851(NP)、AY496852 (M1)。与国内已发表的H9N2亚型禽分离株的NP和M1基因核苷酸序列和氨基酸同源性进行比较，结果表明，NP核苷酸同源性在90.51%-99.73%之间，推导氨基酸同源性在95.38%-99.40%之间；M1核苷酸同源性在90.78%-99.08%之间，推导氨基酸同源性在95.63%-100%之间，表明A型AIV的NP基因及M1基因的高度保守性。分离株MJ00与A/Chicken/GuangDong/11/97(H9N2)同源性最高，亲缘关系最近。其中，NP基因核苷酸同源性为99.73%，仅有4个核苷酸发生变异，氨基酸同源性为99.40%，仅有3个氨基酸发生变异。M1基因核苷酸同源性为99.08%，仅有7个核苷酸发生变异，氨基酸同源性为100%。系统进化树分析表明，分离株NP和M1基因均属于H9N2 AIV欧亚分支的Y280-like亚分支。
将NP基因和M1基因分别插入pGEX-6p-1和PET-30a原核表达载体当中，构建了分别含有NP、M1基因的原核表达载体pGEX-6p-NP、PET-30a-NP、PET-30a-M1，为NP、M1基因的表达及进一步研究AIV NP与宿主特异性的关系、M1基因在病毒感染中的作用及禽流感的诊断和免疫奠定了一定的分子生物学基础。
Avian influenza (AI) was an infection and /or disease syndrome caused by avian influenza A virus (AIV), which was firstly reported in 1878 in Italy. AI had spread all over the world. Poultry and wild fowl both were infected by AIV. AIV had numerous subtypes and its genome had the characteristics of recombinant and high degree of variation, which took difficulty to the prevention and diagnosis of AI. In China, the epidemic AI among poultry was reported in 1994 in Guangdong, AIV H9N2 subtype had been the main virus type, and which had made more danger to chicken. The nucleoprotein (NP) and matrix protein M1 (M1) of AIV were of type specificity and had high conservative sequence, both of which could be the foundation of classification and diagnosis. So we study NP and M1 from molecular level so as to prevent and diagnose AI.
In this research, the AIV（H9N2） isolate was propagated in chicken embryos. The virus was purified by ultracentrifuge. According to the characteristics of AIV genome and the NP gene and M1 gene sequence published by Genbank, a primer T-1 was designed to amplify the cDNA by reverse transcript. Other primers NP-u/NP-1 and M1-u/M1-1 were designed to amplify the NP gene and M1 gene. The PCR product was cloned into pMD18-T vector. The positive recombinant clone was identified by PCR and endonuclease digest. Then the inserting DNA fragment was sequenced and analysed. The NP gene of AIV (H9N2） isolate was 1497bp, which encodes 498 amino acids, M1 gene was 759bp in length, encoding 252 amino acids. The sequences of NP gene and M1 gene had been submitted to Genbank, the number were AY496851(NP) and AY496852(M1). Compared with other published NP and M1 cDNA sequences, it showed that the homology of NP gene was from 90.51% to 99.73% and the homology of amino acids was from 95.38% to 99.40%, the homology of M1 gene was from 90.78% to 99.08% and the homology of amino acids was from 95.63% to 100%. The result showed that the NP gene and M1 gene had high conservative sequence. The result also showed Avian Influenza Virus A/chicken/Mudanjiang/0823/00(H9N2) strain had the close relationship with A/Chicken/Guangdong/11/97(H9N2). The homology of NP gene was 99.73% and the homology of amino acids was 99.40%, only 3 amino acids was different, the homology of M1 gene was from 90.78% to 99.08% and the homology of amino acids was from 95.63% to 100%. The analysis of phylogenetic tree showed that NP and M1 gene of the isolate were Y280-like sublineage of Eurasian avian lineage.
The NP and M1 Gene were cloned into the prokaryotic expression vector pGEX-6p-1 and PET-30a respectively. The recombinant vector was identified by endonuclease. The result showed that the target gene was subcloned successfully into the expression vector and construct the recombinant expression vector pGEX-6p-NP, PET-30a-NP and PET-30a- M1.The expression vectors containing NP and M1 gene facilitates the study for the relationship both NP and host particularity, the function of M1 in virus infection and prevention and diagnosis of AI.
Candidate: Guan Xueting
Major: Preventive Veterinary Medicine
Supervisor: Prof. Wang Junwei
引文
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