Cloning And Expression Of Antigen Eg95 Gene From Echinococcus Granulosus In Xinjiang

In some highly prevalent regions in China,such as Xinjiang, Qinghai, Gansu, Ninxia, etc, there were more than 5%-10% peoples have been infected vaccine by this parasite. Development and application of effective have been being considered as the most efficient way to control this sort of transmission-infected diseases. Eg95 protein was regarded as a vaccine candidate,no information is available as yet concerning genetic variability in the gene encoding Eg95 between E.g strains from New Zealand sheep origin and E.g isolates from Xinjiang sheep origin.The genomic DNA and total RNA was extracted from the protoscolex of E. granulosus in Xinjiang with Universal Genomic DNA Extraction Kit and RNAiso Reagent. The primers were designed according to the parent Eg95 sequence. The Genomic DNA was used as template in PCR amplification with the primers, and then ligated to the pGM-T plasmid. The positive clones of pGM-T/Eg95 were identified by PCR, NcoⅠand PstⅠenzyme digestion and sequencing. ExonⅡwhich is the length of 300bp Gene, was amplified with a pair of specific primers by PCR. The length is about 300bpwas cloned into pGEX-6P-1 plasmid, expressed in E. coli BL21.PartⅠshowed that the total RNA was used as template in PCR amplification with the primers, the PCR fragment is wrong, maybe the postprocessing of sample was unfit And another DNA sequence analysis of Eg95 Xinjiang strain (Eg95-XJ) genomic fragments indicated that the length of Eg95-XJ-1 was 1304bp. By homology comparison, there were 86%～99% homology of Eg95 gene between Eg95-XJ-1 and other Eg95 gene family members of Gene Bank 97%～99% homology of Eg95-XJ-1 gene was found in comparison to that of the Qinghai strain. Although Eg95-XJ-1 was different from them, the multiple nucleotide differences occured predominantly in the non-coding regions of gene. The results show that Eg95-XJ belongs to the same family of Eg95 gene. The results of PartⅡshowed that the specific Eg95 protein was expressed by pGEX-6P-1/Eg95 in the E.coli BL21. The recombination protocaryon expression plasmid of pGEX-6P-1/Eg95 was successfully constructed and could be used as candidate vaccine for further study on immune precaution.