cTnI-linker-TnC融合蛋白的原核表达及鉴定

doi:10.13523/j.cb.20150407

中国生物工程杂志

2015, Vol. 35

Issue (4): 48-53 DOI: 10.13523/j.cb.20150407

研究报告

cTnI-linker-TnC融合蛋白的原核表达及鉴定

龚隆财^1,2, 罗镇明^1,2, 杨雁青^1,2, 王振宇^1,2, 向军俭^1,2, 王宏^1,2

1. 暨南大学广东省分子免疫与抗体工程重点实验室广州 510632;
2. 广州市疾病与食品安全免疫学快速检测重点实验室暨南大学分室广州 510632

Prokaryotic Expression and Identification of cTnI-linker-TnC Fusion Protein

GONG Long-cai^1,2, LUO Zhen-ming^1,2, YANG Yan-qing^1,2, WANG Zhen-yu^1,2, XIANG Jun-jian^1,2, WANG Hong^1,2

1. Department of Bioengineering, Guangdong Province Key Laboratory of Molecular Immunology and Antibody Engineering, Jinan University, Guangzhou 510632, China;
2. Guangzhou Key Laboratory of Disease and Food Safety Rapid Test, Guangzhou 510632, China

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摘要：

从PUBMED中查找人心脏cTnI和cTnC的cDNA序列,在两者之间加上15个中性氨基酸残基(G4S)3的linker编码序列,并分别构建原核表达质粒pET32a-cTnI-linker-TnC和pET28a-cTnI-linker-TnC,转化入大肠杆菌表达菌株BL21(DE3)中,用异丙基硫代-β-D-半乳糖苷(IPTG)诱导其表达。通过SDS-PAGE电泳和间接ELISA对目的蛋白进行初步鉴定,并优化表达条件以实现目的蛋白的可溶性高表达。用标准抗cTnI单克隆抗体和标准抗cTnC单克隆抗体对目的蛋白进行Western blot 鉴定和双抗体夹心ELISA鉴定,同时用八位心肌梗塞患者血清做间接ELISA鉴定。结果表明,cTnI-linker-TnC融合蛋白在表达载体pET32a中实现了可溶性高表达,最佳表达条件为28℃、0.4mmol/L IPTG、诱导表达5h;且融合蛋白具有较好的免疫原性,保存了cTnI和cTnC单体的天然结构,有望成为天然cTnI-TnC复合物的替代物,为下一步制备高特异性的抗cTnI-TnC复合物的单克隆抗体奠定了基础。

关键词： cTnI-linker-TnC; 融合蛋白; 原核表达; 双抗体夹心ELISA

Abstract:

The aim is to express and identify the fusion protein cTnI-linker-TnC, and detect its immunogenicity for preparation of monoclonal antibody of anti-cTnI-TnC. A short DNA sequence which codes 15 neutral amino acid residues was used to link cTnI and cTnC. Then, the recombinant prokaryotic expression vectors pET32a-cTnI-linker-TnC and pET28a-cTnI-linker-TnC were constructed to express the fusion protein cTnI-linker-TnC by transformed into E.coli BL21 cells respectively. And the fusion proteins cTnI-linker-TnC were identified by 12% SDS-PAGE and indirect ELISA test.At the same time, the concentrations of the inducing agent,expression times and temperatures were optimized to obtain high soluble target protein. At last, the commercial monoclonal antibodies of anti-cTnI and anti-cTnC were used to do the Western blot test and double antibody sandwich ELISA,and the serum of myocardial infarction patients were used to do the indirect ELISA. The results showed that the fusion protein cTnI-linker-TnC has a high soluble expression, and has reserved its immunogenicity well. This indicates the fusion protein cTnI-linker-TnC may replace the natural cTnI-TnC compound in the future.

Key words: cTnI-linker-TnC Fusion protein Prokaryotic expression Double antibody sandwich ELISA

收稿日期: 2014-12-30 出版日期: 2015-04-25

ZTFLH:

Q789

基金资助:

广东省战略新兴产业核心技术攻关项目(2012A080800007)资助项目

通讯作者: 王宏 E-mail: wanghonghlj@yahoo.com.cn

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引用本文:

龚隆财, 罗镇明, 杨雁青, 王振宇, 向军俭, 王宏. cTnI-linker-TnC融合蛋白的原核表达及鉴定[J]. 中国生物工程杂志, 2015, 35(4): 48-53.

GONG Long-cai, LUO Zhen-ming, YANG Yan-qing, WANG Zhen-yu, XIANG Jun-jian, WANG Hong. Prokaryotic Expression and Identification of cTnI-linker-TnC Fusion Protein. China Biotechnology, 2015, 35(4): 48-53.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150407 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I4/48

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