Studies On Cloning And Expressing Of Major Antigenic Genes Of Avian Influenza Virus Subtype H9N2 And Mycoplasma Gallisepticum HS Strain

Avian influenza virus (AIV) and Mycoplasma gallisepticum (MG) are two of the most important causes of respiratory diseases that damage the poultry industry heavily and bring huge financial loss. In recent years, AIV have been epidemic in China, especially high pathogenic AIV burned out in China and some other countries in Asia at the beginning of 2004. Both the govements of the world and the ordinary people were focusing on the burst of AIV, not only because of the huge damage to poultry industry in economy but because of the infection and death of the human being. Addionally, MG is one of the most common causes of the respiratory diseases. Its infection rate is very high, often high to more than 50%. Furthermore, MG can be spread by eggs and frequently follow by the secondly affection and leads to heavy damage in poultry. In order to enhance the products of poultry industry and protect the health of people, it is imperative to investigate the traits of the major antigenic gene in molecular biology and tend to contribute to the study of early diagnose and control of the two diseases.In this paper, studies on cloning and expressing of major antigenic genes of avian influenza virus A/chicken/China/HSS2004 (H9N2) strain and MG-HS strain was carried out. First, nucleoprotein (NP) and hemagglutinin (HA) gene was cloned and expressed in E.coli.The methods and results reported as following. (1) NP gene cDNA and HA gene partial cDNA were amplified with the method of reverse transcription polymerization chain reaction (RT-PCR). The cDNAs were cloned into pMD-18T vector and screened positive clones to sequence. (2) The full-length cDNA of NP gene was 1525bp and shared high homology with that of the same avian subtype (97%) or other subtype (more than 90%). The cDNA of HA gene obtained was 1172bp and shared nearly 99% homology with that of the same avian subtype isolated in China, which suggested that H9N2 in China come from the same strain. Analyzing the split site of HA, structure of R-S-S-R ↓ G located near the site which was low pathogen AIV feature. (3) Recombinant plasmid pKG-NP and pKG-HA were constructed to express NP gene and HA gene in E.coli. After Inducing by IPTG, a high efficiency expression of fusion protein GST-NP derived from pKG-NP was obtained in BL21 codon plus. The level of expression was high up to 20%of the total cell proteins of E.coli. Molecular weight of expressed products is approximately 84kD by SDS-PAGE analysis. Results of Western-blotting showed that GST-NP could react to the chicken antiserum against AIV with high immunoiogical competence. While pKG-HA failed to express in BL21 codon plus or BL21 (DE3). (4) Inclusion body of recombinant NP was purified by N-larcosine Na salt (SKL). Through denaturing, annealing, permeating and concentrating, purified recombinant NP was obtained.Second, based on the work of Changjiang Weng and Qingchun Shen, pMGA1.2, one pMGA gene in MgW17, was subcloned by polymerase chain reaction (PCR). The cloned DNA was cloned into prokaryotic vector pGEX-KG and expressed in E.coli BL21 (DE3).The product was near 55kD comprised of 26 kD GST and 243 remainder amino acid. Western-blot suggested the product react with serum of chicken anti MG.Based on these work, three "TGA" were same-sense mutated into "TGG" and got four fragments I , II, Illand IV, the molecule weight were 730bp, 174bp> 387bp and 546bp respectively. After digestion and ligatation, pMGA1.2 including three "TGG" were inserted into pGEX-KG and expressed in E.coli BL21 (DE3). Products approximately 92 kD was detected by SDS-PAGE analysis. The level of expression was high up to 25% of the total cell proteins of E.coli. Western-blot revealed that fusion protein could react with the antibody of Mycoplasma gallisepticum and showed high immunoiogical activity.Additionally, fragments Eland ITJ+IV products were obtained too, which were 39kD and 60kD and showed immunoiogical activity. These studies would provide a useful base for serological diagnosis and vaccine study and prevention of MG infection.In this study, NP gene and HA gene were obtained by RT-PCR. NP gene was expressed in E.coli at high level. At the same time, pMGA1.2 gene was subcloned and in which three TGA encoding Thr were same-sense mutated. The modified pMGA1.2 gene was expressed in E.coli at high level. Recombinant NP and recombinant pMGA provided the patient antigen in high purity and lower cost in the detection of antibody and early rapid diagnose of AIV and MG infection and make it feasible to study on the gene engineer vaccines.