胶原Ⅳα1链NC1结构域基因的克隆及其在大肠杆菌中的表达

 胶原中多个成分如ⅩⅤ,ⅩⅤⅢ的NC结构域能抑制血管内皮细胞增殖和迁移,具有抑制肿瘤生长和转移的活性.从胎盘组织中提取总RNA,根据文献报道的胶原Ⅳα₁链cDNA序列,设计一对特异性引物,应用RT-PCR方法扩增出胶原Ⅳα₁链NC1结构域基因.DNA序列测定发现,扩增得到的基因与文献报道序列存在单个核苷酸变异(A132C),编码氨基酸(Gly)不变.将扩增得到的胶原Ⅳα₁链NC1结构域基因克隆到原核表达载体pPROEXHTb上,并在大肠杆菌BL21中进行体外表达.

Cloning and Expression of Human Collagen IV α1 Chain NC1 Domain

It has been found that many components of collagens such as Collagen Ⅹ Ⅴ,Ⅹ Ⅷ NC domain can inhibit proliferation, migration of vessel endothelial cell as well as suppress tumor growth and metastased. Here a pair are designed if specific primers accordng to the uncleotide sequence of human collagen Ⅳ α₁ chain NC1 domain which reported, then total RNA was extracted from human placenta tissue and human collagen Ⅳ α₁ chain NC1 domain was amplified by RT-PCR. The result from nucleotide sequencing indicated that there is single nucleotide mutation from A to C at position 132 but deduced amino acid(Gly) is not changed. The human collagen Ⅳ α₁ chain NC1 domain was then cloned into prodaryotic expression vector pPROEXHTb and overexpressed in E.coli BL21.