阴道毛滴虫半胱氨酸蛋白酶3基因的克隆、表达与免疫原性分析

中国寄生虫学与寄生虫病杂志 ›› 2009, Vol. 27 ›› Issue (6): 7-487.

阴道毛滴虫半胱氨酸蛋白酶3基因的克隆、表达与免疫原性分析

贾万忠, 李志, 赵亮, 聂芳芳, 伦照荣*

中山大学生命科学学院寄生生物研究中心，广州 510275

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2009-12-30 发布日期:2009-12-30

Cloning, Expression and Immunogenicity Analysis of Cysteine Proteinase 3 of Trichomonas vaginalis

JIA Wan-zhong, LI Zhi, ZHAO Liang, NIE Fang-fang, LUN Zhao-rong*

Center for Parasitic Organisms, State Key Laboratory of Biocontrol, School of Life Sciences, Zhongshang University, Guangzhou 510275，China

Received:1900-01-01 Revised:1900-01-01 Online:2009-12-30 Published:2009-12-30

摘要/Abstract

摘要： 目的研究小鼠对阴道毛滴虫（Trichomonas vaginalis）半胱氨酸蛋白酶3（TvCP3）重组蛋白的免疫应答。方法用PCR方法从阴道毛滴虫基因组DNA扩增TvCP3基因编码序列，分别用编码前体酶和成熟酶的基因片段构建重组表达质粒pET28b-TvCP3和pET28b-TvCP3C，转化入大肠埃希菌（E. coli）BL21（DE3）后进行诱导表达，通过金属螯合层析法（immobilized metal affinity chromatography，IMAC）纯化表达产物重组蛋白，复性后免疫BALB/c小鼠。BALB/c小鼠分为TvCP3免疫组、TvCP3C免疫组和对照组3组，每组6只，分别用TvCP3重组蛋白、TvCP3C和PBS免疫小鼠。第1次25 μg/只，福氏完全佐剂乳化；第2次25 μg/只，福氏不完全佐剂乳化；第3次与第4次12.5 μg/只，水剂。前3次免疫间隔2周，第4次间隔1周。末次免疫后1周用ELISA测定血清抗体滴度。采集高滴度小鼠血清制备免疫血清，通过蛋白质印迹（Western blotting）分析抗体所识别的阴道毛滴虫虫体或其分泌物中的特异性抗原组分。结果重组表达质粒 pET28b-TvCP3和pET28b-TvCP3C均能在E. coli BL21（DE3）中高效表达，重组蛋白占菌体总蛋白的25%以上；ELISA结果显示，纯化的重组蛋白TvCP3和TvCP3C免疫小鼠4次后血清抗体效价分别达1︰204 800和1︰102 400；Western blotting分析显示，小鼠免疫血清能特异性识别表达产物中的目的蛋白，以及阴道毛滴虫虫体或分泌物中的特异性抗原组分。结论重组表达质粒pET28b-Tvcp3和pET28b-Tvcp3C可在E. coli BL21（DE3）中高效表达，纯化的表达产物具有良好的免疫原性。

关键词: 阴道毛滴虫, 半胱氨酸蛋白酶3, 重组抗原, 酶联免疫吸附试验, BALB/c小鼠

Abstract: Objective To study the humoral antibody response of mice to the recombinant Trichomonas vaginalis cysteine proteinase（TvCP3）in order to investigate the function of the proteinase and its application in diagnosis. Methods T. vaginalis cysteine proteinase gene 3（TvCP3）was cloned by using PCR, and was inserted into the expression vector pET28b. The recombinant plasmid pET28b-TvCP3 or pET28b-TvCP3C（a matured and pre-matured enzyme fragment）was then transformed to Escherichia coli BL21（DE3）. The recombinant protein was expressed in E. coli，purified by IMAC（immobilized metal affinity chromatography）, and was used to immune BALB/c mice. The mice were divided into groups TvCP3, TvCP3C and control, 6 in each group. The first and second injections for each mouse were administered with 25 μg purified TvCPs which was emulsified with an equal volume of Freund’s complete and incomplete adjuvants, respectively. Two more injections were done using 12.5 μg purified antigens without adjuvants, with 2 weeks interval between the first three injections and one week interval between the 3rd and 4th injections. The murine serum samples were detected one week post the 4th injection. The specific IgG antibody in the serum against the recombinant protein was evaluated by ELISA and Western blotting. Results The expression level of TvCP3 and TvCP3C reached to more than 25% of the total amount of proteins expressed by the bacteria respectively, and the purity in both of them was more than 80% after purified by cobalt-based IMAC resin. ELISA showed that both purified recombinant TvCP3 and TvCP3C induced a high titer of antibodies in the immunized mice（1/204 800 and 1/102 400, respectively）. Western blotting analysis indicated that the antibodies reacted with a specific band of the whole T. vaginalis antigens or soluble fractions from T. vaginalis cultures. Conclusion The recombinant TvCP3 and TvCP3C proteins have been highly expressed in E. coli BL21（DE3）and the expressed products can induce high level of antibodies in BALB/c mice, which recognized a specific band of proteins from T. vaginalis soluble fractions and cultures in vitro.

Key words: Trichomonas viginalis, Cysteine proteinase 3, Recombinant antigen, ELISA, BALB/c mice

贾万忠;李志;赵亮;聂芳芳;伦照荣. 阴道毛滴虫半胱氨酸蛋白酶3基因的克隆、表达与免疫原性分析[J]. 中国寄生虫学与寄生虫病杂志, 2009, 27(6): 7-487.

JIAWan-zhong;LIZhi;ZHAOLiang;NIEFang-fang;LUNZhao-rong*. Cloning, Expression and Immunogenicity Analysis of Cysteine Proteinase 3 of Trichomonas vaginalis[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2009, 27(6): 7-487.

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