摘要
华支睾吸虫病是由于华支睾吸虫寄生在人体或终宿主的肝胆管内引起的一种重要的人兽共患寄生虫病,不但给渔业带来巨大的经济损失,而且也严重影响人类的健康。华支睾吸虫主要在亚洲国家流行,我国也是感染严重国家,据最近一次全国寄生虫流行病学调查数据显示(2004年公布),全国流行区感染超过1200万。虽然对该病已引起了越来越多的重视,但是对于该寄生虫的功能基因及与宿主的相互作用和人体之间的免疫机制等仍有很多问题没有阐明。构建cDNA文库是大量获得功能基因的好方法,而且也为功能基因的进一步研奠定良好的基础。同时,通过对cDNA表达文库的免疫学筛选,可以有的放矢的获得高反应性基因,为疾病诊断及疫苗研制奠定基础。本研究以吉林流行区月亮泡镇自然感染的狗体内寄生的华支睾吸虫成虫和月亮泡真市场购买的麦穗鱼体内的囊蚴为实验材料,成功构建了成虫和囊蚴的cDNA表达文库,并以自然感染病人血清为探针,对成虫文库进行了免疫学筛选和部分EST测序,筛选到了6个高反应基因,EST测序1000个,获得一个已知序列和156个新基因。这些为该病的诊断、疫苗研制及华支睾吸虫功能基因的研究奠定了基础。
Clonorchiasis is a serious food-borne parasitic zoonosis, which is mainly exhibited as hepatobiliary lesions caused by parasitic Clonorchis sinensis in human bile ducts. Clonorchis sinensis are predominantly distributed in the East and Southeast Asia. There are totally 35,000,000 infected patients in the world, among which 12,000,000 are in China. Although clonorchiasis has gained increasing attentions, the functional genes of Clonorchis sinensis and the immune mechanisms through which it interacts with humans are still unclear. Construction of cDNA expression libraries is a useful tool for discovering functional genes, which lays a foundation for further study of gene functions. In our work, we performed an immunological screening of the cDNA expression library of Clonorchis sinensis and acquired antigen genes that had high reactionogenicity, which laid a foundation for disease diagnosis and the development of vaccines. Moreover, we also carried out EST sequencing and discovered a large number of functional genes.
In this work, the cDNA expression library of Clonorchis sinensis was constructed using classical approaches. Clonorchis sinensis were collected from fresh livers of natural infected dogs. Total RNA from Clonorchis sinensis was extracted using TRIzol? reagent (Invitrogen) according to the manufacturer’s instructions. RNA samples from multiple extractions were mixed and the D260nm/D280nm and D260nm/D230nm values were measured, which were 1.84 and 2.13,respectively. mRNA was isolated using mRNA Extraction Kit (Stratagene) according to the manufacturer’s instructions. The first chains of cDNAs were synthesized by an MMLV-RT reverse transcriptase using mRNA as a template and Oligo (dT) 18 including a restriction site of Xho I as a primer. The second chains were subsequently synthesized by Colon bacillus DNA polymerase I with addition of Colon bacillus RNase H. After end filling by T4 DNA polymerase, cDNAs were connected to EcoR I adaptors. After digested by Xho I, cDNAs with two adhesive ends, which included EcoR I restriction site at the 5’terminal and Xho I restriction site at the 3’terminal, respectively, were obtained. Double-strand cDNAs were subjected to centrichromatography to get rid of the digested adaptor fragments and the cDNAs that were smaller than 400 bp. Ligation of cDNAs to vectors was carried out by incubation overnight at 12°C. Packaged original cDNA library was acquired after incubation for 2 h at 22°C. Five microliters of cDNA library were used in a gradient dilution to get diluted libraries with concentrations of 10-1, 10-2, 10-3 and 10-4 of the original concertration. Ten microliters of each diluted library were mixed with 350μl XL1-blue bacteria and coated on a NZY agar plate containing X-gal and IPTG. The capacity and the recombination rate of the cDNA library were measured. Furthermore, 100 phage plaques were randomly selected and the recombination rates and the sizes of inserted cDNA fragments were determined by PCR. The results showed that the capacity of the original library was 1.5×106 pfu and the recombination rate was 99%. In addition, the sizes of cDNA fragments were between 400 bp and 2000 bp. After capacity measurement, the remnant cDNA library was entirely coated on the agar plates and amplified. The titer of the amplified library was measured by the method of gradient dilution as described above, which is 1.5×1010 pfu/mL. The library was then stored at -80°C with addition of DMSO.
Immunological screening of the constructed cDNA library was then performed. The antibody titers of the serum samples from 100 patients in the epidemic area were measured by ELISA using a soluble antigen from the adults of Clonorchis sinensis. The serum samples that had high titers were used as probes for the immunological screening. Non-specific antibodies in the serum were removed by pseudoscreening. The genes of the antigens that had high reactionogenicity were obtained by immunological screening using the constructed cDNA library. Screening were repeated until monoclonal positive phage plaques were acquired. The positive clones were verified by PCR and sequencing and analyzed by molecular biology softwares and bioinformatics tools. Forty-one positive clones were obtained by a second screening, among which 11 clones were corresponded to a gene that encodes a protein 87% homologous with glycine-2 in Clonorchis sinensis. Twelve clones were corresponded to genes that encode proline-2 family of proteins and have 55-84% homology. Moreover, eight clones were corresponded to a gene that encodes a protein 98% homologous with Cs44 in Clonorchis sinensis. Another eight clones were corresponded to a gene that encodes a protein 39% homologous with an unknown protein in Nematostella vectensis. In addition, one clone was corresponded to a gene encoding a protein that has 32% homology with transcriptional elongation factor, while another was corresponded to a gene encoding a protein that has 52% homology with the CG3446 gene encoding protein in Drosophila. Among these genes, the ones that encode Cs44 antigen, glycine-2 and proline-2 exhibited strong reaction signals during screening. These three genes also have the same signal peptide sequence and all of them are secreted antigens. The constructed cDNA library was further subjected to EST sequencing. After plating and incubation of the amplified library, 1000 phage plaques were selected and sent to the Beijing Huada Gene Company for sequencing. The 1000 EST sequences obtained from sequencing were analyzed and classified according to the gene copy numbers. Totally 157 genes were found from these sequences, among which 135 were single copy genes, 30 were two copy genes, 25 were three copy genes, 10 were four copy genes, 5 were five copy genes, 3 were six copy genes, 4 were seven copy genes, 3 were nine copy genes, 4 were ten copy genes, 2 were thirty-two copy genes, 2 were fifty-five copy genes, 3 were sixty-six copy genes, and 2 were eighty-two copy genes. A repeated sequence was also obtained. After blaster matching in GENBANK, we found that only one gene has been logged in GENBANK and others are all novel genes. Furthermore, we also constructed the cDNA expression library of metacercaria of Clonorchis sinensis using the same approach in constructing the adult library. The capacity of the original cDNA library of metacercaria was 1.33×106 and the recombination rate was 99%. The titer of the amplified library was 9.5×109 pfu/mL and the sizes of fragments were 400-2000 bp.
The present study lays foundations for the investigation of functional genes in Clonorchis sinensis, for further recombination of genes that encode high-reactionogenicity antigens, for antigen recombination through combined gene expression, for the discovery of sensitive and specific methods for immunological detection of Clonorchis sinensis, and for the development of effective vaccines against Clonorchis sinensis.
引文
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