| Avian influenza(AI) is an acute infectious disease caused by avian influenza virus, threaten human health and social stability severely.The highly pathogenic AI violates the avian respiratory system even causes septicaemiaof whole body and is closely related with a high animal mortality.The application of effective vaccine is the valid method of controlling avian influenza.Based on The cross immunity of M2,NP gene of influenza A virus,become the new development trend of Influenza Vaccine.the key-link techniques in novel vaccines of gene engineering are how to strengthen to produce immune protection.HSP70 plays an important role in regulating the immune system,which could unspecifically enhance the innate immune responses,efficiency of Antigen recognition, processing and presenting,promote activation of immune cells including T Lymphocytes and so on.Initiate adaptive immune responses.Thus,it produces anti-tumor and anti-infection immune response.In the study of modern vaccines, HSP70 could also be used as adjuvant of novel vaccines including gene engineering vaccine and so on.In the present,the immunoregulation function and mechanism of Mycobacterium tuberculosis HSP70 has been mastered and used gradually.However, there is still no systemic study on whether the functions between different species origin have the specificity,for multiple biological species origin of HSP70.There is fewer study on mammalian origin HSP as vaccine adjuvant,the relationship between molecule interior structure and immunoregulation function is not clear.In view of these problems,the studies focus on these fields.HSP70 gene in mycobacterium tuberculosis,quail,chicken and rats origin were successfully cloned.The results show that it shares 100%nucleotide homology with the same species.And it shares 98.6%,97.8%and 92.1%nucleotide homology respectively with poultry,cattle,canine,human origin.Consequently,the functions of HSP70 between avian origin,between cattle and canine origin,between human and chicken origin have the strong consistency.The homologies of bacteria(Escherichia coli and Mycobacterium tuberculosis),avian and mammalian origin HSP are all lower than 50%. So we can say,generation,.Function and exerting mechanism of bacteria HSP70 have major difference from avian and mammalian origin HSP70.Differences in amino acid sequences of Molecule interior structure of each species origin HSP70 were mainly distributed in nucleic acid binding domain and the C-terminal region,however, polypeptide binding domain is relatively conservative.MsHSP70 gene was subcloned into palsmid pET28a(+) and pET32a(+) by PCR, the recombinant plasmid was transformed into Escherichia coli BL21(DE3),plysS and Rosetta(DE3),it was expressed as soluble protein under induction of IPTG and purified by Ni-NTA Agarose.The recombinant gene NPe(epitope enrich regions) and extra-domain of M2 protein(M2e) was constructed by PCR and cloned into plasmids pGEX-6P-1 and pET32a(+).The recombinant plasmids were transformed into Escherichia coli BL21(DE3),plysS and Rosetta(DE3) and expressed under induction of IPTG.The fusion protein TRX-NPe·M2e and GST-NPe·M2e were purified by Ni-NTA Agarose. An indirect ELISA method with detection antigen of fuision protein TRX-NP·M2e was initially established.The certain specificity and sensitivity of indirect ELISA analysis coated by fusion protein NPe·M2e was showed by detection of positive sera.Mouse origin HSP70(383-537),HSP70(383-637) and HSP70(1-637) were used to study their adjuvant effect of gene engineering vaccine using M2e as model antigen.The results showed that HSP(383-537),HSP70(383-637) and HSP70(1-637) can all enhance the specific antibody of M2e.The effect of HSP(383-537) was stronger than HSP70(383-637) obviously,especially significantly stronger than HSP70(1-637).Thus,HSP(383-537) of the mouse can be used as molecular adjuvant(vaccine vector) instead of MHSP70 (1-637) and HSP70(383-637).However,the concrete mechanism remained further experimental proof.
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