Expression and Purification of a Novel Influenza Virus Subunit Vaccine
- VernacularTitle:一种新型广谱流感病毒亚单位疫苗的表达与纯化
- Author:
Yi-Ju WEI
;
Hai-Ting LONG
;
Xu YANG
;
Jian-Fang LI
;
Yan-Wei BI
;
Jian-Feng LI
;
Wei-Ming XU
;
- Publication Type:Journal Article
- Keywords:
Influenza virus Subunit vaccine Multiepitope Expression and purification Antigenicity
- From:
China Biotechnology
2006;0(05):-
- CountryChina
- Language:Chinese
-
Abstract:
The influenza A virus matrix protein2 gene(M2)which deleted transmembrane region was amplified by overlap extending PCR,and the multiepitope gene of hemagglutinin(HA)was PCR amplified with seven continuous synthesized segments by designing primer.The two gene segments were separately cloned into pMD18T vector to sequence analysis and prokarytic expression vector pET28a+ to construct the recombinant plasmid pET28a+M2dHA.The recombinant plasmid was transformed into E.coli BL21(DE3),and the high expression strain was obtained by screening monoclones.The recombinant protein existed as inclusion bodies,which accounted about 45% of the total cellular protein.The inclusion bodies were washed with 1% Triton X100 solution twice,and dissolved in 8 mol/L urea solution.The solution protein was purified by Ni+2 affinity chromatography,and refolded by dilution renaturation,then purified by Q Sepharose FF cation exchange column.The purity of the protein was over 90% by HPLC analysis.The result of Western blot showed it has good antigenicity and specificity.These results strongly supported for the further study of the broadspectrum influenza virus subunit vaccine.
