Xu Xue-Nian, Zhou Yan, Dong Yu-Ting, Tan Yu-Guang, Bao Yi-Fang, Xu Bin, Cheng Na, Xu Hong-Bo, Li Xue-Ming, Feng Zheng
National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai 200025, China.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2010 Dec 30;28(6):401-5.
To screen and identify new specific antigen gene from a cDNA library of adult Clonorchis sinensis, and investigate the immunogenicity of the recombinant proteins.
The lambdaZAP 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.
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 PPMP I and type PPMP II antigens, respectively. Sequence analysis revealed that these PPMP genes were a novel specific C. sinensis antigen gene family. Two new genes, PPMP I Cs2 and PPMP II Cs3, 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.
A novel PPMP gene family of C. sinensis has been identified, and its recombinant proteins show high immunogenicity.
从华支睾吸虫成虫cDNA文库中筛选并鉴定新的特异性抗原基因，研究重组蛋白的免疫原性。
用华支睾吸虫病患者混合血清对λZAP华支睾吸虫成虫cDNA文库进行免疫筛选。对阳性克隆进行测序和分析。将编码成熟肽的序列克隆到原核表达载体pET28b(+)中。将重组质粒转化到大肠杆菌BLR21(DE3)或BLR21(DE3)pLysS中，然后用IPTG诱导蛋白表达。重组蛋白用His-bind树脂（Ni-NTA）亲和层析法纯化，并用Western印迹法鉴定。用纯化的重组pET28b-Cs2蛋白免疫BALB/c小鼠，用ELISA分析免疫小鼠血清中的特异性抗体。
从华支睾吸虫cDNA文库中分离出44个阳性克隆。3个含有PPMP氨基酸序列特异性串联重复的克隆被命名为华支睾吸虫PPMP抗原基因。含有KPPMPGDRDA、QPPMPGGRDA的基因分别被命名为PPMP I型和PPMP II型抗原。序列分析表明，这些PPMP基因是一个新的华支睾吸虫特异性抗原基因家族。两个新基因PPMP I Cs2和PPMP II Cs3在大肠杆菌中表达，SDS-PAGE显示这两种重组蛋白的分子量分别约为22 000和39 000。这两种可溶性重组蛋白能被华支睾吸虫病患者混合血清识别。免疫小鼠产生了高水平的针对重组蛋白的特异性IgG（最大稀释度1:64 000）。
已鉴定出一个新的华支睾吸虫PPMP基因家族，其重组蛋白具有高免疫原性。
