| 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 XuetingMajor: Preventive Veterinary MedicineSupervisor: Prof. Wang Junwei...
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