Detection Of The Antibodies Level Of Swine Influenza Virus,Neuraminidasecloning And Initial Construction Of Prokaryotic Expressing Vector

To understand infection situation due to swine influenza virus among pig folks in guangxi province,combined serologic methods including hemagglutination inhibition(HI)test and ELISA test were used to detect the antibodies level of swine influenza virus.To carry out detection of the antibodies of SIV subtypes(H1 and H3),339 and 291 serum samples were collected on boars,sows and piglets respectively in scale pig farms,small pig farms and extensive pig farms from 10 areas of Guangxi,.For 344 tested serum samples using HI and ELISA methods,the results demonstrated that:175 and 195 serum were respectively tested positive with 50.87%(175/344)and 56.68%(195/344) rate using HI and ELISA methods;HI method and ELISA method rate reached 89.74%(175/195).11 mixed scale pig farms were infected with different degree of H1 and H3 SIV subtypes,antibody average rate of H1,H3 and H1+H3 SIV subtypes were 60.5%,42%and 21.65%;but positive rate of pigs serum including H1,H3 and H1+H3 SIV of others piglets from small pig farms and extensive pig farms were relatively lower.In order to detect SIV using RT-PCR assay from 2006 to 2007 year,251 tissue samples(lung,spleen,lymphe nodes)and 155 cotton swabs were respectively collected in 130 suspected pig farms of 14 areas and 8 suspected pig farms of 4 others cities.Results indicated that,72 and 11 samples were respectively positive.That to prove that at present,H1 and H3 subtype swine influenza infections widely existed in the Guangxi pig flocks,and were potential disasters for the pig industry.At the same time,to construct a prokaryotic expressing vector, primer was designed according the nucleotide sequence of neuraminidase(NA),positive samples had been used to amplify NA by RT-PCR assay,RT-PCR product was cloned into PMD-18 vector and sequenced,recombinant PMD-NA plasmid was cloned into PET-32a(+)to construct prokaryotic expressing vector,novel recombinant plasmid of the constructed expressing vector was transformed into BL21 cell medium culture,has been identified by digestion method and sequencage.Results demonstrated that the amplified NA has 1410 bp, homology comparison of others strains nucleotides and amino acid sequences were between 96.1%-99.6%and 96.%-99.4%,sequences were demonstrated to be specific for SIV H1N2 subtype.After identication using RT-PCR and digestion techniques of the recombinant plasmid and the constructed expressing vector recombinant plasmid,results desmontrated that the prokaryotic expression vector was successfully constructed,to be used for the control of swine influenza virus and the basis of vaccine for further research.