Liu Qian, Xu Xue-Nian, Zhou Yan, Cheng Na, Dong Yu-Ting, Zheng Hua-Jun, Zhu Yong-Qiang, Zhu Yong-Qiang
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2013 Aug;31(4):245-50.
To find and clone new antigen genes from the lambda-ZAP cDNA expression library of adult Clonorchis sinensis, and determine the immunological characteristics of the recombinant proteins.
The cDNA expression library of adult C. sinensis was screened by pooled sera of clonorchiasis patients. The sequences of the positive phage clones were compared with the sequences in EST database, and the full-length sequence of the gene (Cs22 gene) was obtained by RT-PCR. cDNA fragments containing 2 and 3 times tandem repeat sequences were generated by jumping PCR. The sequence encoding the mature peptide or the tandem repeat sequence was respectively cloned into the prokaryotic expression vector pET28a (+), and then transformed into E. coli Rosetta DE3 cells for expression. The recombinant proteins (rCs22-2r, rCs22-3r, rCs22M-2r, and rCs22M-3r) were purified by His-bind-resin (Ni-NTA) affinity chromatography. The immunogenicity of rCs22-2r and rCs22-3r was identified by ELISA. To evaluate the immunological diagnostic value of rCs22-2r and rCs22-3r, serum samples from 35 clonorchiasis patients, 31 healthy individuals, 15 schistosomiasis patients, 15 paragonimiasis westermani patients and 13 cysticercosis patients were examined by ELISA. To locate antigenic determinants, the pooled sera of clonorchiasis patients and healthy persons were analyzed for specific antibodies by ELISA with recombinant protein rCs22M-2r and rCs22M-3r containing the tandem repeat sequences.
The full-length sequence of Cs22 antigen gene of C. sinensis was obtained. It contained 13 times tandem repeat sequences of EQQDGDEEGMGGDGGRGKEKGKVEGEDGAGEQKEQA. Bioinformatics analysis indicated that the protein (Cs22) belonged to GPI-anchored proteins family. The recombinant proteins rCs22-2r and rCs22-3r showed a certain level of immunogenicity. The positive rate by ELISA coated with the purified PrCs22-2r and PrCs22-3r for sera of clonorchiasis patients both were 45.7% (16/35), and 3.2% (1/31) for those of healthy persons. There was no cross reaction with sera of schistosomiasis and cysticercosis patients. The cross reaction with sera of paragonimiasis westermani patients was 1/15. The recombinant proteins rCs22M-2r and rCs22M-3r which only contained tandem repeats were specifically recognized by pooled sera of clonorchiasis patients.
The Cs22 antigen gene of Clonorchis sinensis is obtained, and the recombinant proteins have certain diagnostic value. The antigenic determinant is located in tandem repeat sequences.
从华支睾吸虫成虫λ-ZAP cDNA表达文库中寻找并克隆新的抗原基因，确定重组蛋白的免疫学特性。
用华支睾吸虫病患者混合血清筛选华支睾吸虫成虫cDNA表达文库。将阳性噬菌体克隆的序列与EST数据库中的序列进行比较，通过RT-PCR获得该基因（Cs22基因）的全长序列。通过跳跃PCR产生含2倍和3倍串联重复序列的cDNA片段。将编码成熟肽或串联重复序列的序列分别克隆到原核表达载体pET28a(+)中，然后转化到大肠杆菌Rosetta DE3细胞中进行表达。用His-bind-resin（Ni-NTA）亲和层析法纯化重组蛋白（rCs22-2r、rCs22-3r、rCs22M-2r和rCs22M-3r）。用ELISA鉴定rCs22-2r和rCs22-3r的免疫原性。为评估rCs22-2r和rCs22-3r的免疫诊断价值，用ELISA检测35例华支睾吸虫病患者、31例健康个体、15例血吸虫病患者、15例卫氏并殖吸虫病患者和13例囊尾蚴病患者的血清样本。为定位抗原决定簇，用含串联重复序列的重组蛋白rCs22M-2r和rCs22M-3r通过ELISA分析华支睾吸虫病患者和健康人的混合血清中的特异性抗体。
获得了华支睾吸虫Cs22抗原基因的全长序列。它含有13次EQQDGDEEGMGGDGGRGKEKGKVEGEDGAGEQKEQA串联重复序列。生物信息学分析表明该蛋白（Cs22）属于GPI锚定蛋白家族。重组蛋白rCs22-2r和rCs22-3r表现出一定水平的免疫原性。用纯化的PrCs22-2r和PrCs22-3r包被ELISA检测华支睾吸虫病患者血清的阳性率均为45.7%（16/35），健康人血清的阳性率为3.2%（1/31）。与血吸虫病和囊尾蚴病患者血清无交叉反应。与卫氏并殖吸虫病患者血清的交叉反应为1/15。仅含串联重复序列的重组蛋白rCs22M-2r和rCs22M-3r被华支睾吸虫病患者混合血清特异性识别。
获得了华支睾吸虫Cs22抗原基因，重组蛋白具有一定的诊断价值。抗原决定簇位于串联重复序列中。
