Construction Of Recombinant Plasmid Of E.stiedae MIC-5 And Rabbit IL-6 Fusion Gene

Coccidiosis is a kind of protozoan diseases in rabbits caused by coccidian of Eimeriidae,which widely infected to rabbits in our country.The infection rate of Immature rabbits was 100%,the mortality was more than 70%.Clinical symptoms included diarrhea,slow growth,thin.To the etiology survey,E.stiedae can cause serious liver coccidiosis.Now,the main method,which Control rabbit's coccidiosis,is chemical substances,but followed the chemical drug residues and resistance,more and more scholars are seeking immunological methods to prevent and control vaccine.Recently,some scholars use isolated strains of rabbit coccidian to make strong coccidia vaccine.The occidian vaccine would cause pathogen diffusion,because of rabbit coccidia have very species.So,many scholars have development of an effective molecular adjuvant and DNA transport carrier to enhance the immune effect of Nuclear vaccine and their safety.This study had cloned and expressioned the antigen candidate gene of sporulated oocysts E.stiedae MIC-5 gene,cloned rabbit IL-6 gene,sequence analysis;construction of recombinant plasmid Es MIC-5 gene and rabbit IL-6 gene fusion gene.And the fusion gene was subcloned into pGEX-KG and pCDNA3.1(-) vector.last,the mice immunity experiment showed that the rabbit IL-6 gene can enhance Es MIC-5 gene Nuclear vaccine.1 Construction of DNA vaccine pCDNA3.1(-)-Es MIC-5The pure E.stiedae oocysts were floated through saturated salt water and cultured to sporulate under 28℃.The E.stiedae were treatment by Naclo,then put it into homogenizer used the DEPC-treated, They were grinded 30min,when 80%coocysts were broken.Total RNA of the pure E.stiedae was extracted by TRIZOL method,,using RT-PCR amplification Es MIC-5 gene of 1100bp, according to the instruction..And use the same enzyme digested Es mic-5 gene.The PCR products were inserted into the corresponding restriction site in the plasmid pMD18-T.Construction of pMD 18-T-MIC-5.Then this gene were subcloned into the pGEX-KG and pCDNA3.1(-).Next, transformed pGEX-KG-MIC-5 into E.coli BL21,induction culture at 37℃with IPTG,the SDS-PAGE Electrophhoresis analysis of MIC-5 fusion protein indicated that there were about 56Ku.2 Cloning and sequence analysis of rabbit IL-6 geneRabbits IL-6 gene was amplified from spleen by RT-PCR,using specific primers which published in GeneBank,and rabbits IL-6 gene was inserted into the same site of plasmid pMD18-T to generate the plasmid pMD 18-T-IL-6 by using standard cloning procedures.The gene has 726bp longer which include rabbit IL-6 gene of the whole open reading frame.Compared with the European rabbit,the Chinese rabbit IL-6 gene,the homology were 100%.and compared with the different Species for example water buffalo,Wild boar,chicken,Norway Rats,mice,Cattle,pigs,horses of IL-6 gene homology were 60.4%,64.3%,42.7%,54.3%,53.9%,61.2%,64.5%,65.4%%.It improved that different species of the IL-6 gene have a big difference.3 Construction of Recombinant plasmid of MIC-5-IL-6 fusion geneEs MIC-5 gene was amplified by RT-PCR,using specific primers with EcoR I/site in the forward and Sal I site in the downward,and the EcoR I/ Sal I-digested fragment was inserted into the EcoR I/Sal I site of plasmid pMD18-T to generate the plasmid pMD18-T-IL-6.At the same time, rabbits IL-6 gene was amplified,and the PCR fragment of Es MIC-5 digested by and Sal I was inserted into the same site of the plasmid pMD 18-T-IL-6'to generate the fusion gene MIC-5-IL-6.At last the EcoR I/ HindⅢ-digested MIC-5-IL-6 fragment was inserted into to pGEX-KG and pCDNA3.1(+) vector.Through the dual-enzyme digestion,it improved that the fusion gene has been cloned in prokaryotic expression and eukaryotic expression vector.So transformed pGEX-KG MIC-5-IL-6 into E.coli BL21,and induction culture at 37℃with IPTG,it showed that the MIC-5-IL-6 fusion protein molecular was about 85Ku by the SDS-PAGE analysis.32 mice were randomly divided into 4 groups.Interval 15 days in three times to immunized. The I group was injected with DNA vaccine of pCDNA3.1(-)-MIC-5-IL-6,Ⅱgroup was injected with DNA vaccine of pCDNA3.1(-)-MIC-5,Ⅲgroup was immunized with empty vector,Ⅳgroup was the control group without any treatment.The ELISA test results:There were significant differences betweenⅠgroup andⅡgroup(P＜0.05) in the two immunized.Third immunized,Ⅰgroup was significantly higher than theⅡgroup(P＜0.01).