人B7-1基因与GPI信号肽序列融和基因的原核表达载体的构建及表达

Construction and expression of procaryotic expression vector of a chimeric GPI-B7-1 cDNA

目的构建GP1锚定蛋白基因与B7—1分子融合的原核高效表达载体（GPI—B7—1），观察融合蛋白的抗肿瘤效应。方法分别从新鲜胎盘和ConA刺激的外周血单核细胞中克隆人胎盘碱性磷酸酶（hPLAP-1）C末端信号肽序列（GPI）和CD80（B7—1）基因。利用PCR技术将GPI和B7—1胞外编码区基因进行融合，并将融合基因亚克隆入原核表达载体（pET-30a）中。重组载体pET-30a—GPI—B7—1转化表达菌株E．coli BL，纯化GPI—B7—1融合蛋白，SDS—PAGE、Western blot分析鉴定其免疫活性。结果PCR和RT—PCR产物经电泳鉴定，在100～250bp处可见到133bp的GPI目的条带。在750bp左右可见到792bp的B7-1胞外编码区基因目的条带。重组载体pET-30a—GPI-B7—1经PCR检测获得900bp左右的目的片段，经EcoRI、Sall双酶切鉴定，得到5000bp和900bp左右大小2个片段，实现了融合蛋白（GPI—B7—1）在大肠杆菌中的高效表达，在非变性条件下纯化该融合蛋白，SDS—PAGE分析显示，表达蛋白的分子量约为38kDa，Western blotting证实38kDa处出现一条特异的显色带，该融合蛋白即为目的蛋白。结论GPI—B7—1表达载体可在大肠杆菌中高效表达获取融合蛋白，为肿瘤免疫治疗奠定了基础。

Objective Through constructing prokaryotic expression vector pET-30a-GPI-B7-1, to gain purified GPI-B7-1 fusion protein so as to confirm the tumor immune effect. Methods The DNA fragment encoding the signal for GPI-anehor attachment of hPLAP-1 and the eDNA encoding the human eostimulatory molecule CD80 （ B7-1 ） were cloned from fresh placenta and human peripheral blood monocytes （PBMC） respectively. The two fragment were annealed to form a fusion gene （GPI-B7-1） by PCR. Then the fusion gene was inserted into the prokaryotic expression vector pET-30a, resulting in pET-30a-GPI-BT-1. Transfer to E. coli BL21, purified fusion protein were analysed by SDS-PAGE and Western blot. Results Agarose gel electrophoresis map of GPI and B7-1 PCR products show that GPI goal gene strap was seen at 133bp region and B7-1 goal gene strap at 792 region. Identification of recombinant pET-30a-GPI-B7- 1 by restriction enzyme and PCR illustrate two goal fragment for 5000 bp and 900 bp, to realize the expression of fusion gene （ GPI-B7-1 ） at the E. coli BL21. The fusion protein was successfully produced in the pET expression system induced by IPTG and purified by Ni2 ＋ -NTA agarose column. By SDS-PAGE and Western blot analysis, the observed molecular weight of the fusion protein was 38 kDa. Conclusion The purified GPI-B7-1 fusion protein can be obtained from E. cofi BL21 transfered by prokaryotic expression vector pET-30a-GPI-B7-1, which will prove useful tool for the study of tumor immune therapy.