牛金属硫蛋白素bMTcin编码区的克隆及其在大肠杆菌中的表达研究

Cloning and Expression in E. Coli of the Coding Region for Bovine Metallothioneincin

以本实验室由RT—PCR扩增得到的含有牛金属硫蛋白(bMT)cDNA的重组质粒pYJ101为模板,PCR扩增出牛金属硫蛋白素bMTcin的编码区,将其克隆到pGEM—T载体上并进行DNA序列测定。利用融合表达载体pGEX4T—1,在E.coli中诱导表达bMTcin,经过Glutathine Sepharose 4B亲和层析纯化后,SDS—PAGE检测GST:bMTcin的条带。结果表明,所克隆的bMTcin序列全长为141bp,其DNA序列与原模板中的序列完全相同,与GenBank中另外5个bMTcin DNA相比,第31位和86位与Conneely的序列为A和G,导致第11位(Thr)和29位(Arg)的氨基酸不同于其他4个序列(11位为Ala,29位为Lys)。融合表达产物亲和层析纯化后,用SDS-PAGE检测可见融合蛋白GST:bMTcin的条带,紫外分光光度计测定,融合蛋白GST:bMTcin的表达量大约为1.1mg/L发酵液。

The coding region for bMTcin was obtained by PCR amplification of the plasmid pYJ101, which contains the whole bMT cDNA by RT - PCR amplification, and then cloned into pGEM - T vector. bMTcin sequence was determined, which consists of 141bp, its sequence was same as original template, Comparision with five other bMTcin DNA sequences registered in GenBank shows 100% homology with Conneely in DNA sequence, However, there are two base substitutions (nucleotide 31 G→A, nucleotide 86 A→G) with four other bMTcin DNA, they lead to two amino acids change (residue 11 Thr→Ala, residue 29 Arg→Lys). After fusion expression product was purify by affinity chromatograghy, we saw the band corresponding to fusion product GST: bMTcin on SDS-PAGE. The expression level of fusion protein GST:bMTcin is approximately 1. 1 mg/L culture, determined by ultraviolet spectrophotometry.