HCMV-UL138 CTL表位肽重组酵母菌的构建及其免疫效应

doi:10.3969/j.issn.2095-9400.2015.06.004

科研成果详情

题名	HCMV-UL138 CTL表位肽重组酵母菌的构建及其免疫效应
其他题名	Construction and immune response of recombinant whole-cell yeast expressing HCMV-UL138 CTL epit-ope
作者	张跃进1; 柳献云2; 陈向敏3; 田晓娟 4; 李文姝4; 薛向阳4
发表日期	2015-06-25
发表期刊	温州医科大学学报影响因子和分区
语种	中文
原始文献类型	学术期刊
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
其他关键词	human cytomegalovirus (HCMV) ; CTL epitope ; HBV surface antigen (HBsAg) ; recombinant yeast
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、NetCTL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(Pre S2)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/Pre S2-HBsAg-UL13815-27重组质粒。重组质粒经Bgl I处理线性化后,电转化至GS115菌株中,构建Pre S2-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Ig G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/Pre S2-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Pre S2-HBsAg-UL13815-27灭活全菌体免疫小鼠后,可检测到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Pre S2-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。
其他摘要	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 expres-sion. The constructed pPIC3.5K/PreS2-HBsAg-UL13815-27 recombinant plasmid was linearized by Bgl II restric-tion enzyme and electricity transformed into GS115 strain to construct PreS2-HBsAg-UL13815-27 recombinant yeast. Identiifed recombinant strains were then induced by methanol. The expression of aim protein was identi-ifed by Western Blot and ELISA methods using HBsAg antibodies. The identiifed recombinant yeast cells were inactivated by heat and vaccinated mice by subcutaneous injection. Speciifc 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 in-duced by the methanol, HBsAg-speciifc antibodies conifrmed the expression of PreS2-HBsAg-UL13815-27 with molecular weight about 33 kD in the lysate of the recombinant yeast. The immunization of inactivated recombi-nant whole-cell yeast expressing PreS2-HBsAg-UL13815-27 could induce signiifcantly anti-HBsAg-speciifc 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 signiifcantly increased in the group of inactivated re-combinant 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 speciifc UL138-speciifc cellular immune response can be induced in the immunized mice with recombinant whole yeast cells.
资助项目	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）
ISSN	2095-9400
卷号	45 期号:06 页码:406-412
DOI	10.3969/j.issn.2095-9400.2015.06.004
页数	7
收录类别	CNKI ; 万方 ; 维普 ; ISTIC
URL	查看原文
社科自定义期刊分类	G类
引用统计
文献类型	期刊论文
条目标识符	https://kms.wmu.edu.cn/handle/3ETUA0LF/49118
专题	护理学院基础医学院（机能实验教学中心）附属第一医院检验医学院（生命科学学院、生物学实验教学中心）_生物学实验教学中心
作者单位	1.温州医科大学护理学院; 2.温州医科大学附属第一医院中药房; 3.温州医科大学生物学实验教学中心; 4.温州医科大学分子病毒与疫苗研究所微生物与免疫学教研室
第一作者单位	护理学院
第一作者的第一单位	护理学院
推荐引用方式 GB/T 7714	张跃进,柳献云,陈向敏,等. HCMV-UL138 CTL表位肽重组酵母菌的构建及其免疫效应[J]. 温州医科大学学报,2015,45(06):406-412.
APA	张跃进, 柳献云, 陈向敏, 田晓娟, 李文姝, & 薛向阳. (2015). HCMV-UL138 CTL表位肽重组酵母菌的构建及其免疫效应. 温州医科大学学报, 45(06), 406-412.
MLA	张跃进,et al."HCMV-UL138 CTL表位肽重组酵母菌的构建及其免疫效应".温州医科大学学报 45.06(2015):406-412.