Construction of recombinant yeast expressing PreS2120-146 and hepatitis B surface antigen and evaluation of the immune effects of whole yeast cells
10.3760/cma.j.issn.1000-6680.2014.11.004
- VernacularTitle:表达乙型肝炎表面抗原及前 S2蛋白120～146片段重组酵母菌的构建及全菌体免疫效应评价
- Author:
Xiangmin CHEN
;
Yuejin ZHANG
;
Xiaojuan TIAN
;
Ping XIA
;
Weiwen PAN
;
Tian XIA
;
Chenhui YU
;
Lifang ZHANG
;
Xiangyang XUE
- Publication Type:Journal Article
- Keywords:
Hepatitis B surface antigens;
PreS2;
Pichia;
Immune responses
- From:
Chinese Journal of Infectious Diseases
2014;(11):660-665
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the recombinant yeast expressing PreS2 120-146-hepatitis B surface antigen (HBsAg),and to evaluate the immune effects of whole yeast cells.Methods PreS2 120-146 and HBsAg gene sequence were optimized according to the yeast cell codon preference,and were recombined and cloned into pPIC3.5K yeast expression vector to construct pPIC3.5K/PreS2 120-146 plasmid.After digested and linearized by Bgk Ⅱ restriction enzyme,pPIC3.5K/PreS2 120-146-HBsAg recombinant plasmid was electrotransformed into GS115 strain to screen PreS2 120-146-HBsAg-recombinant Pichiapastoris .The expression of PreS2 120-146-HBsAg was identified by sodium doclecyl sulfate polyacrylamide gel electrophogesis (SDS-PAGE),Western blot and enzyme linked immunosorbent assay (ELISA)analysis. BALB/c mice were vaccinated by inactivated whole recombinant yeast cells expressing target protein. Specific antibodies to HBsAg were detected by ELISA.Cytotoxic T lymphocyte (CTL)response induced by interferon (IFN)-γ was detected by reverse transcription-polymerase chain reaction (RT-PCR)when immune spleen cells of mice were stimulated by CTL epitope on HBsAg.Independent sample t test was used. Results Data of PCR detection,restriction enzyme digestion and sequencing analysis showed that recombinant pPIC3.5K/PreS2 120-146-HBsAg plasmid was successfully constructed.SDS-PAGE,Western blot and ELISA verified the expression of PreS2 120-146-HBsAg in the lysate of the recombinant Pichiapastoris induced by methanol.Levels of specific anti-HBsAg IgG antibodies produced by inactivated yeast cells vaccinated mice were comparable to purified HBsAg immunization (t =0.946,P =0.381 ). Analysis of HBsAg-specific CTL responses revealed that the level of IFN-γwas significantly higher when the immune spleen cells of mice were stimulated by CTL epitope peptides on HBsAg (t =2.305 ,P =0.044).Conclusions PreS2 120-146-HBsAg target protein is successfully expressed by construction of recombinant Pichiapastoris . The specific humoral and cellular immune responses are induced by recombinant whole yeast cells vaccinated mice.
