Tropic1808基因的原核表达及其表达产物的生物活性

Tropic180 8基因是新近获得的一个鼠源性的cDNA .Tropic180 8基因开放阅读框架片段通过PCR方法从质粒中扩增后 ,重组入表达载体pET 2 1a中 ,转化大肠杆菌BL2 1(DE3 ) .用IPTG诱导目的蛋白的表达 ,SDS PAGE并凝胶图象分析确定目的蛋白表达水平占细菌总蛋白的 14%以上 .表达蛋白在N端融合有 16个氨基酸 ,将表达蛋白电转移至PVDF膜 .氨基酸序列分析表明 ,其N端第 17～ 2 5位氨基酸序列与Tropic180 8基因编码序列一致 .利用融合部分含T7·Tag ,通过亲和层析纯化表达蛋白 ,经Westernblot检测为目的蛋白 ,加入到无血清培养的新生SD大鼠背根神经节 (DRG)中 ,观察到表达蛋白对DRG具有促进存活和促进突起生长的作用 .

Expression of the Tropic 1808 Gene in Escherichia coli and Bioactivity of Expression Product

Tropic1808 gene is a novel cDNA acquired from rats. Opening reading frame of Tropic 1808 gene was amplified by PCR and cloned into the pET 21a vector, then transformed into Escherichia coli BL21(DE3) and induced by IPTG for the expression of recombinant protein. By SDS PAGE and image analysis, the recombinant product accounted for more than 14% of the total bacterial proteins. The expression protein, fused with 16 amino acids to N terminus, was transferred to PVDF membrane. Amino acid sequence analysis showed that 17 to 25 amino acid sequences of N terminus were consistent with Tropic 1808 gene. Fused with T7·Tag , the expression protein was purified by affinity chromatography and detected by Western blot. Co culture with dor sal root ganglion of neonatal SD rats in serum free showed that the purified protein promote DRG cell survival and neurite outgrowth. Results provided basis for preparing corresponding antibody,and researching its structure,functions and clinical applications.