家蚕抗菌肽CM4和人可溶性BAFF融合基因的克隆、表达及活性鉴定

Cloning Expression and Characterization of a Bi-functional Antibacterial Peptides CM4 and hsBAFF Fusion Protein in Escherichia Coli

采用PCR法从含有人可溶性BAFF基因的pET30a(+)扩增得到人sBAFF基因,通过rPCR将抗菌肽Cecropin CM4基因的2条单核苷酸链进行扩增得到CM4基因,再采用over-lap PCR法通过linker将hsBAFF与Cecropin CM4融合基因相连接.经纯化和鉴定后,定向插入到原核表达载体pET30a(+)中,然后转化E.coliBL21(DE3),通过实验确定了表达该融合基因的最佳诱导条件:IPTG终浓度为1.0mmol/L,诱导时间为5h,温度为30℃,其表达量占全菌蛋白的40%.表达产物经SDS-PAGE分析,得到相对分子质量约为22000的重组蛋白并且存在超声裂解后的上清中.重组蛋白经Western blot检测,结果显示重组蛋白可被鼠抗人可溶性BAFF的抗体识别.采用分子筛Sephadex G-75对重组融合蛋白进行纯化,并经SDS-PAGE对其鉴定.通过对其生物学功能的检测得知,纯化后的重组融合蛋白对大肠杆菌K12D31和真菌有明显的抑菌能力.

The cDNA of human soluble B lymphocyte stimulator （hsBAFF） was amplified by PCR from pET30a （ ＋ ） and the gene of antibacterial peptide CM4 by rPCR. The fusion gene of hsBAFF and CM4 was amplified by using overlap PCR. The prokaryotic expression plasmid pET30a（ ＋ ）/hsBAFF - CM4 was constructed with recombinant DNA techniques after purifying and identifying the DNA fragment. Then the plasmid pET30a（ ＋ ）/hsBAFF -CM4 was transformed into BL21 （DE3） cells and the expression was optimized with proper inducing conditions of 1.0mmol/L IPTG, 5 h and 30℃ induction. The expression level of the target fusion protein reached 40 % of the total bacterial protein. The results of SDS - PAGE indicated that molecular weight of the expressed protein was about 22 000 and the expressed protein mainly existed in the supernatant after sonication. Western blot analysis proved that the recombinant protein has good reactive ability against mouse anti-human soluble BAFF IgG. The expression product was punrified by Sephadex G -75. The purified recombination protein displayed antimicrobial activity obviously.