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. 2004 Oct 15;10(20):2989-93.
doi: 10.3748/wjg.v10.i20.2989.

Yeast expression and DNA immunization of hepatitis B virus S gene with second-loop deletion of alpha determinant region

Hui Hu  1 , Xiao-Mou Peng, Yang-Su Huang, Lin Gu, Qi-Feng Xie, Zhi-Liang Gao
Affiliations

Yeast expression and DNA immunization of hepatitis B virus S gene with second-loop deletion of alpha determinant region

Hui Hu et al. World J Gastroenterol. .

Abstract

Aim: Immune escape mutations of HBV often occur in the dominant epitope, the second-loop of the a determinant of hepatitis B surface antigen (HBsAg). To let the hosts respond to the subdominant epitopes in HBsAg may be an effective way to decrease the prevalence of immune escape mutants. For this reason, a man-made clone of HBV S gene with the second-loop deletion was constructed. Its antigenicity was evaluated by yeast expression analysis and DNA immunization in mice.

Methods: HBV S gene with deleted second-loop, amino acids from 139 to 145, was generated using splicing by overlap extension. HBV deleted S gene was then cloned into the yeast expression vector pPIC9 and the mammalian expression vector pcDNA3 to generate pHB-SDY and pHB-SD, respectively. The complete S gene was cloned into the same vectors as controls. The deleted recombinant HBsAg expressed in yeasts was detected using Abbott IMx HBsAg test kits, enzyme-linked immunoadsorbent assay (ELISA) and immune dot blotting to evaluate its antigenicity in vitro. The anti-HBs responses to DNA immunization in BALB/c mice were detected using Abbott IMx AUSAB test kits to evaluate the antigenicity of that recombinant protein in vivo.

Results: Both deleted and complete HBsAg were successfully expressed in yeasts. They were intracellular expressions. The deleted HBsAg could not be detected by ELISA, in which the monoclonal anti-HBs against the alpha determinant was used, but could be detected by Abbott IMx and immune dot blotting, in which multiple monoclonal anti-HBs and polyclonal anti-HBs were used, respectively. The activity of the deleted HBsAg detected by Abbott IMx was much lower than that of complete HBsAg (the ratio of sample value/cut off value, 106+/-26.7 vs 1 814.4+/-776.3, P<0.01, t = 5.02). The anti-HBs response of pHB-SD to DNA immunization was lower than that of complete HBV S gene vector pHB (the positive rate 2/10 vs 6/10, 4.56+/-3.52 mIU/mL vs 27.60+/-17.3 mIU/mL, P = 0.02, t = 2.7).

Conclusions: HBsAg with deleted second-loop of the alpha determinant still has antigenicity, and can also raise weak anti-HBs response in mice to DNA immunization, suggesting that it is possible to develop a subdominant vaccine for preventing infections of immune escape mutants of HBV.

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Figures

Figure 1
Figure 1
Electrophoresis of the fragments of HBV S gene. Lane 1: HB fragment of complete HBV S gene; Lane 2: HB-SD fragment with the second-loop deletion of the α determinant; Lane 3: DNA fragment of HBV S gene from codon 1 to codon 138; Lane 4: DNA fragment of HBV S gene from codon 146 to codon 226; Lane 5: HBY fragments; Lane 6: HB-SDY fragments; Lane M1: DNA marker.
Figure 2
Figure 2
RFLP analysis of recombinant vectors of pHB-SD and pHB-SDY. Lane M1: DNA marker; Lane 1: HB-SD fragment; Lane 2: pHB-SD candidate digested by restriction endonu-clease Hind III and Eco RI; Lane 3: pHB-SD candidate digested by restriction endonuclease Bam HI; Lane 4: pcDNA3 digested by restriction endonuclease Bam HI; Lane 5: pHB-SDY candi-date digested by restriction endonuclease Not I and Eco RI; Lane M2: DNA marker.
Figure 3
Figure 3
Screening integrants and SDS-PAGE analysis. A: Screening integrants using PCR with AOX1 primers. Lane M2: DNA marker; Lane 1: PCR products of Mut+ transformant without gene of interest; Lane 2: PCR products of Muts transformants with pHBY; Lane 3: PCR products of Muts transformants with pHB-SDY; Lane 4: PCR products of pPIC9 alone. Lane M1 were DNA markers. B: Coomassie-stained SDS-PAGE analysis of the recombinant proteins of pHB-SDY and pHBY. Lane M3: protein marker; Lane 5: cell lysate super-natants of yeast alone; Lane 6: yeast transformed with pPIC9; Lane 7: yeast transformed with pHBY; Lane 8: yeast trans-formed with pHB-SDY.
Figure 4
Figure 4
Antigenicity analysis of recombinant proteins using ELISA and immune dot blotting assay. (A) ELISA, (B) immune dot blotting assay.
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