CAJ | 학술논문

目的构建GPI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100～250 bp处可见到133 bp的GPI目的条带.在750 bp左右可见到792 bp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900 bp左右的目的片段,经EcoRⅠ、SAlⅠ双酶切鉴定,得到5000 bp和900 bp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38 kDa,Western blotting证实38 kDa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.
목적 구건GPI묘정단백기인여B7-1분자융합적원핵고효표체재체(GPI-B7-1),관찰융합단백적항종류효응.방법 분별종신선태반화ConA자격적외주혈단핵세포중극륭인태반감성린산매(hPLAP-1)C말단신호태서렬(GPI)화CD80(B7-1)기인.이용PCR기술장GPI화B7-1포외편마구기인진행융합,병장융합기인아극륭입원핵표체재체(pET-30a)중.중조재체pET-30a-GPI-B7-1전화표체균주E.coli BL,순화GPI-B7-1융합단백,SDS-PAGE、Western blot분석감정기면역활성.결과 PCR화RT-PCR산물경전영감정,재100～250 bp처가견도133 bp적GPI목적 조대.재750 bp좌우가견도792 bp적B7-1포외편마구기인목적 조대.중조재체pET-30a-GPI-B7-1경PCR검측획득900 bp좌우적목적 편단,경EcoRⅠ、SAlⅠ쌍매절감정,득도5000 bp화900 bp좌우대소2개편단,실현료융합단백(GPI-B7-1)재대장간균중적고효표체,재비변성조건하순화해융합단백,SDS-PAGE분석현시,표체단백적분자량약위38 kDa,Western blotting증실38 kDa처출현일조특이적현색대,해융합단백즉위목적 단백.결론 GPI-B7-1표체재체가재대장간균중고효표체획취융합단백,위종류면역치료전정료기출.
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-anchor attachment of hPLAP-1 and the cDNA encoding the human costimulatory molecule CD80 ( BT-1 ) were cloned from fresh placenta and human peripheral blood monocytes (PBMC) respectively. The two fragment were annealed to form a fusion gene (GPI-BT-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 BT-1 PCR products show that GPI goal gene strap was seen at 133bp region and BT-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. coli BL21 transfered by prokaryotic expression vector pET-30a-GPI-B7-1, which will prove useful tool for the study of tumor immune therapy.