淡紫拟青霉丝氨酸蛋白酶基因克隆表达及抗血清的制备

目的]探索利用大肠杆菌进行丝氨酸蛋白酶PL基因的克隆与表达及抗血清制备的方法。方法]根据报道的PL蛋白基因序列设计1对引物,通过RT-PCR扩增获得PL基因片段,对重组载体进行构建、鉴定和序列分析,用表达的特异蛋白条带制备抗原,免疫家兔制备抗血清。结果]通过RT-PCR扩增得到长约850 bp的PL基因片段,将其克隆到原核表达载体pET-22b(+)中,获得的重组子pET-22b-PL转化E.coliBL21(DE3),经最终浓度为1 mmol/L IPTG诱导37℃培养4 h,获得约31 kDa大小的重组蛋白。SDS-PAGE电泳分析表明,该蛋白表达量占菌体总蛋白的60%,说明该蛋白得到了高效表达。用表达的特异蛋白条带制备的免疫家兔制备抗血清,经ELISA测定效价为1/10 000。结论]通过基因重组可获得PL基因在大肠杆菌中的高效表达蛋白,且该蛋白具有较高的免疫活性。

Cloning and Expression of PL gene from Paecilomyces lilacinus and Its Antiserum Preparation

Objective] The propose was to explore the methods for using Escherichia coli to make the cloning and expression of PL gene from the fungus of Paecilomyces lilacinus and to make the antiserum preparation.Method] A pair of primers was designed according to the PL gene sequence that had reported.The PL gene fragment was obtained through RT-PCR amplification and the recombinant vector was conducted and identified and then its sequence was analyzed.The expressed special protein brands were used to prepare the antigen which prepared the anti-serum through immunizing rabbit.Result] A PL gene fragment with length of 850bp was obtained by RT-PCR amplification and it was cloned into the pET-22b(+) to get recombinant vector which was transformed into E.coli BL21(DE3).The target fusion peptide with molecular weight of 31 kDa was obtained under the condition of induction by IPTG at final concentration of 1 mmol/L at 37 ℃ and culture for 4 h.SDS-PAGE electrophoresis analysis showed that its maxim expression amount accounted for 60% of total soluble proteins,indicating that this protein was expressed effectively.Rabbit was immunized using the expressed target peptide as antigen and the antiserum was obtained.The detection of ELISA shows that the antibody potency was 1/10 000.Conclusion] The fusion protein of PL gene that could be highly expressed in Escherichia coli was obtained through the gene recombinant and it had higher immunocompetence.