华支睾吸虫PPMP型抗原基因的克隆、表达与免疫原性鉴定

Cloning,Expression of PPMP Antigen Genes of Clonorchis sinensis and Immunogenic Identification of the Recombinant Proteins

目的筛选华支睾吸虫成虫cDNA表达文库,寻找和鉴别新颖的抗原基因,并分析其重组蛋白的免疫原性方法以华支睾吸虫病患者混合血清免疫筛选华支睾吸虫成虫λZAP cDNA表达文库,将阳性噬菌体克隆、测序,对获得的核苷酸序列进行生物信息学分析。将目的基因成熟肽的编码区克隆至原核表达质粒pET28b(+),转化至大肠埃希菌BLR(DE3),异丙基-β-D硫代半乳糖苷(IPTG)诱导表达。用组氨酸标签亲和纯化柱(Ni-NTA树脂)纯化表达产物,蛋白质印迹(Western blotting)分析其免疫原性。以纯化的重组蛋白pET28b-Cs2免疫BALB/c小鼠,制备免疫血清,ELISA检测抗体水平。结果共获得44个阳性克隆,其中3个克隆的氨基酸序列中均含有PPMP重复序列,将其命名为华支睾吸虫PPMP型抗原,将其中含有KPPMPGDRDA、QPPMPGGRDA串联重复多肽序列的抗原分别称为PPMPⅠ型和PPMPⅡ型抗原。经核苷酸序列同源性比较,PPMP型抗原是一个新的华支睾吸虫特异性抗原家族。PPMPⅠ型的Cs2基因和PPMPⅡ型的Cs3基因的成熟肽编码区在大肠埃希菌BLR(DE3)或BLR(DE3)pLysS中表达,并获得可溶性重组蛋白,2个重组蛋白的相对分子质量分别为M_r 22 000和M_r 39 000。重组蛋白可被华支睾吸虫病患者血清所识别。ELISA结果显示,免疫小鼠可产生高滴度(1:64000)特异性IgG抗体。结论发现华支睾吸虫PPMP型抗原基因家族,其重组蛋白具有较强的免疫原性。

Objective To screen and identify new specific：antigen gene from a cDNA library of adult Clonorchis sinensis,and investigate the immunogenicity of the recombinant proteins.Methods TheλZAP cDNA library of adult C.sinensis was immunoscreened with pooled sera of clonorchiasis patients.The positive clones were sequenced and analyzed. The sequence encoding the mature peptide was cloned into prokaryotic expression vector pET28b（＋）.The recombinant plasmid was transformed into E.coli BLR21 （DE3）or BLR21 （DE3）pLysS and followed by expression of the protein induced by IPTG.The recombinant protein was purified by His-bind-resin （Ni-NTA）affinity chromatography and identified by Western blotting.BALB/c mice were immunized with purified recombinant pET28b-Cs2 protein,and the sera from immunized mice were analyzed for specific antibodies by ELISA.Results A total of 44 positive clones were isolated from the C.sinensis cDNA library.Three clones containing specific tandem repeats of PPMP amino acid sequence were named as C.sinensis PPMP antigen genes.The genes containing KPPMPGDRDA,QPPMPGGRDA were named as type PPMPⅠand type PPMPⅡantigens, respectively.Sequence analysis revealed that these PPMP genes were a novel specific C.sinensis antigen gene family. Two new genes,PPMPⅠCs2 and PPMPⅡCs3,were expressed in E.coli,and SDS-PAGE showed that the two recombinant proteins were about M_r 22 000 and M_r 39 000.The two soluble recombinant proteins were recognized by pooled sera of clonorchiasis patients.A high level of specific IgG against the recombinant proteins （maximum dilution 1：64 000）was produced in immunized mice.Conclusion A novel PPMP gene family of C.sinensis has been identified,and its recombinant proteins show high immunogenicity.