HCMV-UL138 CTL表位肽重组酵母菌的构建及其免疫效应

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Abstract Objective: To construct the recombinant yeast expressing HCMV-UL138 CTL epitope based on the carrier of hepatitis B virus surface antigen (HBsAg), and further explore its immune response. Methods: The complete amino acid sequence of HCMV UL138 was retrieved from the SwissProt Database. The CTL epitopes-riched peptide screened by the methods of SYFPEITHI, Net-CTL and HLA-Bind were inserted into the C-terminal of PreS21-9 sequence, then linked to the N-terminal of HBsAg sequence. The aim sequence optimized according to the yeast cell codon preference was synthesized and cloned into pPIC3.5K vector of yeast expression. The constructed pPIC3.5K/PreS2-HBsAg-UL13815-27 recombinant plasmid was linearized by Bgl II restriction enzyme and electricity transformed into GS115 strain to construct PreS2-HBsAg-UL13815-27 recombinant yeast. Identified recombinant strains were then induced by methanol. The expression of aim protein was identified by Western Blot and ELISA methods using HBsAg antibodies. The identified recombinant yeast cells were inactivated by heat and vaccinated mice by subcutaneous injection. Specific antibodies to HBsAg were detected by ELISA, and cytotoxic T lymphocyte (CTL) response were detected by investigating the interferon-γ (IFN-γ) expression by RT-PCR when immune spleen cells of mice were stimulated by UL13815-27 CTL epitope peptide (VMLVLIVAILCYL). Results: Data of PCR detection, restriction enzyme digestion and sequencing analysis showed that pPIC3.5K/PreS2-HBsAg-UL13815-27 recombinant plasmid was successfully constructed. After induced by the methanol, HBsAg-specific antibodies confirmed the expression of PreS2-HBsAg-UL13815-27 with molecular weight about 33 kD in the lysate of the recombinant yeast. The immunization of inactivated recombinant whole-cell yeast expressing PreS2-HBsAg-UL13815-27 could induce significantly anti-HBsAg-specific IgG antibodies. When the immune spleen cells of mice were stimulated by UL13815-27 CTL epitope peptide, analysis of cellular immune revealed that the production of IFN-γ significantly increased in the group of inactivated recombinant whole-cell yeast expressing PreS2-HBsAg-UL13815-27 than that in the control groups. Conclusion: The recombinant Pichia pastoris yeast expressing PreS2-HBsAg-UL13815-27 protein is successfully constructed. The specific UL138-specific cellular immune response can be induced in the immunized mice with recombinant whole yeast cells.

Key words： human cytomegalovirus (HCMV) CTL epitope HBV surface antigen (HBsAg) recombinant yeast

Received: 02 February 2015

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	ZHANG Yuejin1
	LIU Xianyun2
	CHEN Xiangmin3
	TIAN Xiaojuan4
	LI Wenshu4
	XUE Xiangyang4

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https://xb.wmu.edu.cn/EN/ OR https://xb.wmu.edu.cn/EN/Y2015/V45/I6/406

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