Construction And Immunological Screening Of The CDNA Library Of Metacercaria Of Clonorchis Sinensis

Clonorchis sinensis is one of parasites which have a typical life style of androgynism trematodes. Clonorchiasis is also known as liver fluke disease. Clonorchiasis is caused because a person intakes fishes suffering from Clonorchis sinensis. Clonorchis sinensis adult parasitises in the human bile duct, which will cause mainly hepatobiliary disease. The disease is mainly distributed in Southeast Asia, including China, Korea, Vietnam and other countries. Currently, there are estimated totally 35 million patients who infected with Clonorchis sinensis all over the worldwide region, of which 15 million patients distribute in China, particularly predominantly distributed in the south and northeast China. At present, China has become one of most serious disease against clonorchiasis countries. But so far there have not been obtained satisfactory methods which response to the disease diagnosis and control. Continuing upward trend of infection rates is evident. Besides of an effective vaccine against the disease is not developed which is concerned with the condition. Simultaneously, the ideal diagnostic measures have not been found for helping controlling diseases and cutting off the transmission pathway. Thus study of metacercariae of Clonorchis sinensis functional antigen-specific genes will play a role of significance of early diagnosis and prevention clonorchiasis.In this study, the cDNA expression library of metacercariae of Clonorchis sinensis was constructed using classical Molecular Biology approaches. Kinds of fishes which were infected with metacercariae of Clonorchis sinensis were collected from six different locations of the ponds of Zhenlai Baicheng of Jilin province in China, at the same time, metacercariaes of Clonorchis sinensis were isolated from these infected fishes, then total RNA from metacercariaes of Clonorchis sinensis was extracted by using TRIzol reagent(Invitrogen) according to the manufacturer,s instructions. Subsequently,λZAP Express cDNA Synthesis Kit was used for constructing cDNA expression library of metacercariae of Clonorchis sinensis, and a high-quality cDNA expression library was constructed successfully. Specific procedure was as follows: total RNA from metacercariae of Clonorchis sinensis was extracted using Trizol Reagent(Invitrogen), according to the manufacturer , s introductions. RNA samples from multiple extractions were mixed and the OD260nm/OD280nm and OD260nm/OD230nm values were measured, which were 1.85 and 2.20, respectively. mRNA was isolated using Ploy(A) Quik mRNA Isolation Kit(Stratagene) according to the manufacturer,s instructions. The first strands cDNAs were synthesized by an StrataScript RT reverse transcriptase using mRNA as a template and Oligo(dT) 18 including a restriction site of Xho I as a primer. The second strands were subsequently synthesized by DNA polymerase I with addition of RNase H. After end blunting by Pfu DNA polymerase, cDNAs were connected to EcoR I adaptors. After digested by Xho I, cDNAs with two cohesive terminuses were obtained. CHROMA SPIN-400 column chromatography was used and subjected to centrichromatography to get rid of the digested apaptor fragments and the cDNAs that were smaller than 400bp. Ligation of cDNAs with theλZAP Express vector was obtained after incubation overnight at 12℃. After in vitro packaging, metacercaria of Clonorchis sinensis cDNA expression library was obtained. The parameters of construction of cDNA expression library contained: the quantity of total RNA was 142.17μg, the yield of RNA was 0.66μg/mg, the absorbance of OD260nm/OD280nm was 1.85, OD260nm/OD230nm was 2.20, which indicates high purity of the extracted RNA was acquired; through 1% agarose Gel electrophoresis, total RNA bands were clear, which indicate extraction of total RNA was not degradation. The production of mRNA which was purified was 2.23μg, the yield of mRNA was 1.57%. The capacity of cDNA expression library was 9.15×105pfu, the recombination rate was 99.5%, the sizes of inserted fragments were between the length of 0.4 and of 2.0Kb. Titer of amplified library was 1.6×1010pfu/mL. The library was then stored at 4℃with noting or -80℃with addition of DMSO or 50% Glycerol.Immunological screening of the constructed metacercariae of Clonorchis sinensis cDNA library was then performed. First of all, preparation of a suitable antibody probe for immunoscreening of the cDNA library was substantial, and prepared antibody probe should be treated by adsorption. The procedure of preparation of specific antibody probe was as follows: the most serious affected areas of Clonorchis sinensis infection from the Jilin Province where metacercariaes of Clonorchis sinensis were collected from infected fishes, and isolated from muscle of infected fishes. The isolated metacercariaes of Clonorchis sinensis were used for infecting Japanese white rabbits by mouth. After the procedure of infection, by setting a series of different days and intervals of infection in order to collect serum, and then indirect ELISA was used to test antibodies titer of rabbit serum that was collected from different days and would observe the growth and decline of antibodies titer, from which was selected a suitable antibody probe for immunological screening. In this study, the 14th day rabbit serum has been selected, and this serum was collected from infected rabbits by metacercaria of Clonorchis sinensis. The serum for antibody probe must be treated by pseudoscreening in order to removal of non-specific interference of serum components, at this time, the serum can be used for immunoscreening conducted metacercaria of Clonorchis sinensis cDNA library. Ultimately, high reactogenicity antigen genes of the period of metacercaria of Clonorchis sinensis can be obtained. Immunoscreening procedure was divided into primary and secondary screening, after two rounds of immunoscreening, fifteen positive clones were obtained from metacercaria of Clonorchis sinensis cDNA library by a second screening, among which four clones were corresponded to a gene that encodes a protein highly homologous with Cs44 antigen gene in Clonorchis sinensis. Three clones were corresponded to a gene that encodes a protein which own homologous with glycine-2 gene. And three clones were corresponded to a gene is homologous with the gene which encode proline-2. Maybe further screening procedure will be proceeded. In order to the establishment of sensitive, specific immunological detection and diagnosis methods for the early stages of clonorchiasis, as well as the foundation of vaccine development and function genes research. The further study on the target gene were obtained by immunoscreening will be expressed through a prokaryotic expression system in vitro for a large number of protein.