Study On N And M Gene Of Avian Infectious Bronchitis Virus HN99 Strain

Avian infectious bronchitis(IB) is an acute and highly contagious disease caused by avian infectious bronchitis virus(IBV). IBV most commonly affects the respiratory system, genitourinary system and digestive system in chicken with symptom including dyspnea, egg production drop, nephritis and proventriculitis, etc. IBV is characterized by frequent variations and complex serotypes , which lead to great differences in the clinical manifestations. Diagnostic methods of molecular biology have become popular in recent years since conventional ones are limited. Researches were mainly focused on gene S1,yet late studies indicated that other structural proteins of IBV also have important biological functions. Recently IB epidemicity is trending up in our country,especially kidney-IB and proventriculus-IB which have spread to many areas and caused huge economic loss. It is common sense that the emergence of so many mutant IBV strains is connected with the nonstandard use of live vaccine. Moreover, the data concerning the gene sequences of IBV local epidemic strains in China are not sufficient, and related molecular biological research is not thorough enough. In order to explore the reasons for changeable IBV serotypes and develop the diagnostic reagents in genetic engineering,this study was accomplished on domestic IBV HN99 strain the nucleocapsid gene (N gene) cloning, sequencing, eukaryotic expression and immunological activity detecting of the expression products. Meanwhile membrane gene (M gene) of IBV HN99 strain of domestic isolates were cloned ,sequenced and prokaryotic expression. The results of the research will play a certain role to facilitate the diagnosis and prevention of IB.1. A pair of special primers was designed and synthesized according to avian infectious bronchitis virus N gene sequences published in GenBank. N gene of IBV HN99 strain was amplified by reverse transcription-polymerase chain reaction(RT-PCR). The PCR product was cloned into pMD-18T vector, then transformed into E.coli Top10 competent cells. The pMD-18T-N recombinant plasmid was extracted and the positive plasmid was sequenced after identification by digestion of restriction enzyme and PCR. The results showed that cDNA of N gene of HN99 was composed of 1230 nucleotides encoding a polypeptide of 409 amino acids. Nucleotide homogeneity of N gene of HN99 with N gene of other IBV strains H52,Ark99,BJ ranged from 88.4%-90.7%, while 91.7%-94.4% identity was found on amino acid level. Evolution analysis indicated that HN99 was far related to the other IBV strains such as H52,Ark99,BJ. These results suggested that IBV HN99 was a new variant of IBV.2. M genes of IBV HN99 strain and W strain were amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced. The two M gene sequences, together with their deduced amino acids sequences, were compared with other IBV isolates in the Genbank. With gene insert and gene deletion, M genes of IBV HN99 strain were composed of 669 bp nucleotides encoding 222 amino acids and showed 87.0%-90.3% nucleotide identity and 89.7%-94.2% deduced amino acids identity compared with the M genes of the GDS14, M41, Gray, H52, BJ. With gene insert, M gene of IBV W strain were composed of 681 bp nucleotides encoding 226 amino acids and showed 88.2%-92.7% nucleotide identity and 91.1%-95.6% deduced amino acids identity compared with the M genes of the HN99, M41, Gray, H52, BJ. Phylogenetic analysis revealed that the W strain is closely related to the BJ strain while the HN99 isolate is far related to the other strains of IBV.3. The PCR product of M gene from HN99 strain was digested with EcoRâ… and Hindâ…¢,then cloned into eukaryotic expression vector pF-MBP. The pF-MBP/M were transfected into DH10BAC-eGFP and the recombinant plasmids, named Bacmid-egfp-MBP/M, were transfected into Sf9 cells by liposome. Passage for two generations, the refraction of the transfected Sf9 cells was more evident than normal cells, and the nucleus became bigger. Specific bright green fluorescence was observed across the plasmas. The IBV DNA were detected from pathological Sf9 cells by PCR with primer M13 and specificity primer respectively, substantiating the existence of recombined virus. And the expression of M gene was confirmed by SDS-PAGE and Western Blot. The result showed that the recombined product was 68 ku, which was identical with the expected result.4. The PCR product of N gene from HN99 strain was subcloned into prokaryotic expression vector E.coli pMAL-p2X, pMAL-c2X and PET28a and fused to MBP tag at N-terminus.Then the recombineant plasmid pMAL-p2X-N was transformed into E.coli TB1 and induced by IPTG. Nucleocapsid proteins were purified by affinity chromatography taking advantage of the affinity of MBP for maltose. These purified N proteins were used to raise polyclonal antibody according to classic procedure. SDS-PAGE and Western blot results indicated that MBP-N fusion protein has been successful expressed. Two kinds of protein were appeared after expression, 92 ku and 82ku respectively. Purification product of MBP-N fusion protein cleavage by Factor Xa, MBP tag were cleaved, 45 ku and 35 ku were N proteins. ELLISA confirmed that antiserum to N protein could react with different strains of IBVs, and was more active when it reacted with its relative strains. In conclusion, the non-denaturalized N proteins are active and could be further used as a kind of gene engineering antigen for a group of IBVs diagnosis.To sum up, the results suggested that there existed variability among N gene and M gene of IBV HN99 strains. IBV HN99 strain was far related to the other strains of IBV as a new variant. N gene of HN99 strains was successful expressed in the prokaryotic system. The non-denaturalized N protein had a high biological activity and could be further used as a kind of gene engineering antigen for a group of IBVs diagnosis. M gene of IBV HN99 strain was successful expressed in the eukaryotic system with green fluorescent tag. Gene insert occurred in M gene of the IBV W strain, and its sequence varied. The research revealed the reason for spread and variation order of IBV in China, as well as the expression specificity for the main structural gene on molecular level. The results provided the molecular epidemiological data of IBV for better control and prevention of IB.