重组凝乳酶原基因的原核表达及其抗血清的制备

Prokaryotic expression of recombinant prochymosin gene and its antiserum preparation

目的:原核细胞表达密码子优化的凝乳酶原并制备抗血清。方法:以大肠杆菌密码子的偏爱性整合牛、羊和骆驼的凝乳酶原(pCHY)序列后分别克隆至大肠杆菌原核表达载体pET-30a和真核高效抗体制备核酸疫苗载体pcDNA3-AAT-COMP-C3d3(pcD-ACC)上。原核表达质粒pET30a-pCHY转入大肠杆菌后经IPTG的诱导得到高表达的凝乳酶原,作为检测抗原。真核表达质粒pcDNA3-AAT-COMP-pCHY-C3d3(pACCC)用水动力转染法验证其在mRNA水平的表达后,采用DNA prime-protein boost的策略免疫新西兰大白兔后制备抗体并检测其特异性和效价。结果:SDS-PAGE和Western blot检测表明,成功表达出与预期相对分子质量大小相符的重组凝乳酶原;ELISA检测表明获得高效价的凝乳酶原抗血清。结论:通过密码子优化及核酸疫苗载体pcD-ACC联合DNAprime-protein boost的免疫策略可制备高表达量的重组凝乳酶原,得到接近于蛋白免疫水平的高滴度抗体。

AIM： To optimize the prochymosin （pCHY） gene codons and express the gone in Escherichia coli （ E. coli）, and to prepare its antiserum and detect chymosin protein specifically. METHODS： According to codon usage bias of E. coli, prochymosin gone sequence was synthesized based on the conserved sequences of prochymosin gene from bovine, lamb and camel, and then cloned into the plasmid pET-30a and pcDNA3-AAT-COMP-C3d3 （pcD-ACC）, respectively, pET-30a-pCHY was expressed, as the detected antigen, in E. coli BL21 （ DE3 ） after IPTG induction. RT-PCR was used to detect prochymosin mRNA expression in liver from the mice injected pcDNA3-AAT- COMP-pCHY-C3d3 （pACCC） by hydrodynamics-based transfection method. To prepare the antiserum of prochymosin, pACCC and GST-pCHY proteins were used to immunize New Zealand rabbits in accordance with DNA primeprotein boost strategy. Antibody levels were tested by ELISA. RESULTS： Western blotting showed the molecular weight of His-pCHY protein was about 55 000, similar to the expected molecular size. ELISA demonstrated that the titer level of prochymosin antiserum was high. CONCLUSION： Based on the codon optimization, we have obtained high- titer prochymosin antiserum through DNA vaccine vector pcD-ACC combined with DNA prime-protein boost strategy, similar to that by protein vaccine.