摘要
目的:研究膜型、分泌型TNF-α的31位氨基酸在其生物学效应中的作用,进一步探讨TNF-α结构与生物学效应的关系。方法:采用重叠PCR方法将wt TNF-α31位精氨酸(R)密码子(CGC)定点突变替换成亮氨酸(L)密码子(CTC),构建R31L TNF-α-pcDNA3.0重组质粒,经酶切、PCR及DNA测序鉴定后,采用脂质体转染法将其瞬时转染COS-7细胞进行表达,通过MTT法检测R31LTNF-α突变体的胞毒效应。结果:酶切、PCR及测序鉴定证实目的基因R31LTNF-α正确连接到pcDNA3.0的多克隆位点;TNF-α31位突变可增强sTNF-α的胞毒效应,突变体的CC50值是野生型的1/10,而对mTNF-α的生物学效应无明显影响。结论:本实验成功地构建了R31L TNF-α-pcDNA3.0重组质粒,且在真核细胞COS-7中获得表达;TNF-α31位置换成亮氨酸后,主要增强其分泌型分子的胞毒效应,而对其膜分子无作用。
Objective：To study the role of position 31 Arginine in the amino acid sequnence of two types of TNF-α in its biological function and to investigate the relationship between the structure and biological function of TNF-α.Methods： R31L TNF-α gene was amplified with recombinant ploymerase chain reaction （PCR） by site-direeted mutation of the codon of Arginine （CGC） to that of Leucine （CTC）at position,and the recombinant plasmid R31L TNF-aα-pcDNA3.0 was constructed, which was identified with PCR,double enzyme digestion and DNA sequencing. Then R31L TNF-α-poDNA3.0 was transient-transfected into COS-7 cells with lipofeetamine. After the expression of the mutant, we detected its cytotoxicity with MTT method.Results： The recombinant plasmid R31L TNF-α-pcDNA3.0 was confirmed with PCR, double enzyme digestion and DNA sequencing. The cytotoxicity of R31L sTNF-α was enhanced than that of wt sTNF-α, and the CC50 of R31L sTNF-α is 1/10 of that of wt sTNF-α; but the cytotoxicity of R31L mTNF-α was not different with that of wt mTNF-α. Conduslon： The recombinant plasmid R31L TNF-α-poDNA3.0 was successfully constructed and expressed in CO5-7 cells. The substitution of 31 position amino acid residue of TNF-α to Leucine, enhanced the cytotoxicity of sTNF-α, but did not affect that of mTNF-α.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2008年第11期967-971,975,共6页
Chinese Journal of Immunology
基金
国家自然科学基金资助项目(No.30670421)
