豆状囊尾蚴丝氨酸蛋白酶抑制剂(serpin)的多抗制备、组织分布及活性研究

Polyclonal antibody preparation, tissue localization and activity of serine protease inhibitor serpin of Cysticercus pisiformis

目的研究豆状囊尾蚴丝氨酸蛋白酶抑制剂(Cpserpin)的组织分布及其对丝氨酸蛋白酶的抑制效果。方法利用原核表达的重组Cpserpin (rCpserpin)免疫家兔,采用间接ELISA检测免疫后血清的效价,取免疫兔血清,制备抗体并纯化后,采用Western blotting对多克隆抗体进行特异性分析,通过免疫组化试验分别观察rCpserpin在豆状带绦虫成虫和幼虫中的组织分布。构建重组质粒pPIC9K-Cpserpin,线性化后转化至毕赤酵母KM71感受态细胞,用G418筛选的高拷贝菌株进行诱导表达并纯化rCpserpin,采用发色底物法检测不同浓度(6、9、 18、 21μg/ml) rCpserpin对0.5μg/mlα-糜蛋白酶、胰蛋白酶和弹性蛋白酶等3种丝氨酸蛋白酶活性的抑制效果。结果间接ELISA检测结果显示,免疫血清多抗IgG抗体的效价达1∶51 200。Western blotting分析结果显示,纯化后的多克隆抗体可与rCpserpin和兔豆状囊尾蚴虫体可溶性抗原产生特异性反应。免疫组化试验结果显示,Cpserpin在囊尾蚴阶段表达量很低,但在成节和孕节中高度表达,广泛分布于虫体体壁和虫卵中。酵母表达的rCpserpin随浓度增加对α-糜蛋白酶、胰蛋白酶和弹性蛋白酶的抑制效果分别增强,在21μg/ml浓度时,对3种丝氨酸蛋白酶的抑制率最高,分别为(50.5±2.5)%、(71.5±1.5)%和(77.4±1.5)%(均P <0.05)。结论 rCpserpin多克隆抗体具有特异性,在豆状囊尾蚴成节和孕节中高表达,且其对α-糜蛋白酶、胰蛋白酶和弹性蛋白酶有较强的抑制作用。

Objective To study the tissue distribution of serine protease inhibitor of Cysticercus pisiformis(Cpserpin)and its inhibitory effect on serine proteases.Methods Prokaryotically expressed recombinant Cpserpin(rCpserpin)was used to immunize rabbits,and the titers of rabbit serum after immunization were determined by indirect ELISA.The polyclonal antibodies were purified and analyzed for the specificity by Western blotting.The tissue distributions of rCpserpin in C.pisiformis adult and larva were assayed by immunohistochemistry.The recombinant plasmid pPIC9 K-Cpserpin was constructed,transformed into Pichia pastoris KM71 competent cells after linearization,induced for expression in high-copy strains obtained by G418 screening,and then the products were purified.The purified recombinant protein was tested using chromogenic substrate method for its inhibitory effects on the activity of three serine proteases includingα-chymotrypsin,trypsin and elastase,at the concentrations of 6,9,18 and 21μg/ml,respectively.Results The results of indirect ELISA showed that the titer of serum polyclonal antibody IgG reached 1∶51200.Western blotting showed that the purified polyclonal antibody could specifically react with rCpserpin and the C.pisiformis worm soluble antigen.Immunohistochemistry showed that Cpserpin was expressed at a low level during the cysticercus stage,but was highly expressed in mature proglottid and gravid proglottid,with wide distribution in the worm body wall and eggs.The rCpserpin expressed by P.pastoris showed enhanced inhibitiory effect on the three n of serine proteases with increased concentration,having highest inhibition rate at 21μg/ml forα-chymotrypsin(50.5±2.5)%,trypsin(71.5±1.5)%and elastase(77.4±1.5)%(P<0.05).Conclusion The polyclonal antibody produced displays specificity against rCpserpin,the Cpserpin is highly expressed in mature proglottid and gravid proglottid of C.pisiformis,and the recombinant Cpserpin exhibits strong inhibitory effect onα-chymotrypsin,trypsin,and elastase.