Cloning, Prokaryotic Expression And Application Of Em95 Gene From Echinococcus Multilocularis

Multilocular hydatid disease is also called alveolar echinococcosis(AE), which is a human parasitic zoonosis caused by the larval stage of Echinococcus multilocularis(Alveococcus). Its adult stage parasitizes in the small intestine of foxes, wolves and dogs. The prevalence and distribution of AE is mainly in the high latitudes and frozen tundra in Northern hemisphere. As the growth in international trade and intensified globalization, E. multilocularis seems to be on the increase and the disease now is expending towards Southern hemisphere. It is not only a serious population health problem but also an economic importance globally. Now AE cure mainly depends on surgical treatment and medication as a complementary, but the growth of E. multilocularis larvae is tumor-like and they can metastasize to other visceral organs. The prevention and control of AE has become one of the local governments’ priorities in endemic regions. EM95(the protein is coded by E. multilocularis 95 gene), a candidate protective antigen of E. multilocularis plays a great role in developing vaccines against AE.Em95 nucleotide sequence download from NCBI was used as reference sequence for designing primers and then was amplified with RT-PCR from total RNA as the template. We used bioinformatic softwares for analyzing the ORF of Em95 and found that the length of coding gene for EM95 is 471 bp. EM95 was a secreted protein, with two glycosylate sites, a 16-residue signal peptide in N-terminal and a 23-residue trans-membrane region. The analysis of B-cell eptitopes indicated that the EM95 has 6 potential antigen eptitopes, which is less than EG95. The complete Em95 and the truncated Em95(t Em95, without signal peptide and trans-membrane region fragments) were cloned into the prokaryotic expression vector pET-30 a, respectively and then transformed into E. coli Rosetta and get the destined protein after induced by IPTG. Recombinant protein pET-30a-Em95 was expressed as inclusion body protein and pET-30a-tEm95 was expressed as soluble protein. To evaluate the protection of purified protein, the purified soluble protein was used to immunize the BALB/c mice. The mice(female, 6 weeks old) were divided into three groups: the first group was immunized by 50 μg(25 μg/mL) recombinant protein in Freund’s adjuvant(40 mice); the second group was injected with 200 μL Freund’s adjuvant(40 mice); the third group was as control with 200 μL phosphate buffered saline(40 mice). One month later the mice were injected with half dosage and gave a booster at the second month. After two weeks, each was challenged with 100 protoscolices(peritoneal injection) and was autopsied post 9 weeks. The result showed that the protection rate of recombinant tEM95 protein was 27.58% and the reduced weight of hydatid cyts was 60.52%-67.38%. The result of animal experiment confirmed that the EM95 has a potential to anti-AE.