HCMV-UL138 CTL表位肽重组酵母菌的构建及其免疫效应

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摘要目的：以乙肝病毒表面抗原为载体，制备含人巨细胞病毒（HCMV）-UL138 CTL表位肽的重组酵母菌并分析其免疫效应。方法：根据蛋白质数据库获得HCMV-UL138氨基酸序列，综合应用SYFPEITHI、Net-CTL及HLA-Bind方法筛选富含CTL表位的UL138多肽，插入到乙肝表面抗原前S2（PreS2）1-9区末端再与HBsAg序列连接，在不改变氨基酸密码的前提下，根据酵母偏爱密码子优化序列，进行全序列合成，克隆入pPIC3.5K酵母载体，构建pPIC3.5K/PreS2-HBsAg-UL13815-27重组质粒。重组质粒经Bgl II处理线性化后，电转化至GS115菌株中，构建PreS2-HBsAg-UL13815-27重组酵母，阳性整合菌株经甲醇诱导后，通过Western blot及ELISA方法鉴定目的蛋白表达。鉴定的重组酵母菌经灭活后，全菌体皮下免疫BALB/c小鼠，通过ELISA法检测HBsAg特异性血清IgG抗体；UL13815-27 CTL表位肽（VMLVLIVAILCYL）刺激免疫小鼠的脾细胞，通过检测IFN-γ表达分析诱导产生的CTL反应。结果：PCR、酶切和测序分析表明成功构建了pPIC3.5K/PreS2-HBsAg-UL13815-27重组质粒。重组酵母经甲醇诱导后，酵母裂解液中可检测到HBsAg特异性抗体识别的、分子量约33 kD目的蛋白。表达PreS2-HBsAg-UL13815-27灭活全菌体免疫小鼠后，可检测HBsAg特异性抗体，免疫小鼠脾细胞经UL13815-27 CTL表位肽刺激，IFN-γ表达水平比对照组明显升高。结论：重组酵母全菌体成功表达了PreS2-HBsAg-UL13815-27重组蛋白，且重组酵母菌全菌体免疫小鼠可诱导产生UL13815-27特异性的细胞免疫。

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	张跃进1
	柳献云2
	陈向敏3
	田晓娟4
	李文姝4
	薛向阳4

关键词 ：人类巨细胞病毒, CTL表位, 乙肝病毒表面抗原, 重组酵母

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

收稿日期: 2015-02-02

基金资助:国家自然科学基金资助项目（81001343、81472308）；浙江省自然科学基金资助项目（Y2100909）；温州市科技局科研基金资助项目（Y20120159）。

通讯作者: 薛向阳，副教授，Email：wzxxy001@163.com。

作者简介: 张跃进（1960-），女，浙江温州人，实验师。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2015/V45/I6/406

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