摘要
采用重叠PCR方法,将人绒毛膜促性腺激素β亚基(hCGβ)cDNA3'端与绵羊垂体促性腺激素共有α亚基(oLHα)cDNA 5'端串联起来,构建了hCGβ-oLHα嵌合cDNA。将嵌合cDNA克隆入核型多角体病毒(AcNPV)表达载体pVL1393得到表达型质粒pVL1393-hCGβ-oLHα,并与BaculoGold^(TM)线性化AcNPV基因组DNA共转染昆虫细胞Sf9,筛选得到重组病毒AcNPV-hCGβ-oLHα。以扩增后的重组病毒感染昆虫细胞进行表达,并进一步经偶联抗hCGβ单抗亲和层析柱纯化得到hCGβ-oLHα单链多肽。SDS-PAGE银染和免疫印迹分析结果表明表达产物在非还原条件下表观分子量约40.5kD,还原条件下约为38.0kD,这说明表达产物具有链内二硫链及次级构象。竞争抑制^(125)I-hCGβ与抗体结合的检测结果表明其与hCGβ抗体结合活性较天然hCG弱,但较天然hCGβ略有增加。由此可见单链多肽作为抗hCG夏合抗原避孕疫苗的靶抗原,具有潜在的应用前景。
hCGβ-oLHα chimeric cDNA was constructed by using overlapping PCR to contact the codons of C-terminal end of hCGβ with the codons of N-terminal end of oLHα, then it was subcloned into nuclear polyhedrosis virus (AcNPV) expression vector pVL1393 to construct expression vector pVL1393-hCGβ-oLHα. The insect cells ( Sf9 ) were cotransfected by the expression vector pVL1393-hCGβ-oLHα and BaculoGoldTM AcNPV linearized genomic DNA, and recombinant viruses AcNPV-hCGβ-oLHα were screened out by plaque assay. Further the insect cells were infected by the recombinant viruses, the recombinant hCGβ-oLHα was purified by immunoaffinity chromatography column coupling anti-hCGβ monoclonal antibody from the conditioned media
of infected cells. The results of SDS-PAGE silver staining and western blotting showed that hCGβ-oLHα single peptide chain had apparent molecular weights of 40. 5kD and 38.0kD under non-reducing and reducing conditions respectively, indicating the occurrence of disulfide bonds and significant tertiary structure in the single peptide chain. From the results of competitive inhibition of125I-hCGβ binding we can conclude that the anti-hCGβ antibody-binding activity of hCGβ-oLHα chimera is lower than that of native hCG, but higher than that of native hCGβ. Therefore, we assume that the hCGβ-oLHα chimera should have potential application as a target antigen of anti-hCG fertility regulatory vaccine.
出处
《实验生物学报》
CSCD
1998年第1期61-68,共8页
Acta Biologiae Experimentalis Sinica
基金
国家自然科学基金
国家高科技项目基金~~
关键词
HCG
垂体促性腺激素
单链多肽
生物合成
hCGβ-oLHα chimeric cDNA. Overlapping PCR. Recombinant virus. Insect cell Sf9.Expression.
