The Construction Of Recombinant Expression Plasmid PcDNA3-3-1E Of Eimeria Acervulina

Chicken coccidiosis caused by Eimeria is a worldwide parasitic diseases which dose greatly harmful against chicken and cause huge economic losses to the poultry industry. At present to prevent and treat the disease, chemical-based pharmaceuticals and antibiotics are meninly used.But it is easy to produce drug-resistant.As a result of long-term applications and the poultry product of drug-resistant will affect the human being health.Considering these reasons,all the researchers take their eyes on the vaccine′s receading,and using molecular biology methods has potental priotonty.In this study Eimeria acervulina(E.acervulina) of chicken is the rearch,and the recombinant expression plasmid pcDNA3-3-1E against 3-1E gene of the second generation merozoite is constructed of the surface antigen.The recombinant plasmid is taken as a DNA vaccine for the immunology experiment,It built solid foundation for the development of effective and safe vaccines of anticoccodial as well as new drugs.Based on the sequence alignment of the 3-1E gene of E.acervulina US strain,two primers were designed and synthesized. Using the total RNA of merozoites of E.acervulina Baoding strain isolated from Hebei province of China as template, a partial segment of 3-1E gene was amplified by RT-PCR.The gene fragment 689bp in length was cloned into pGM-T Easy vector, and the recombinant plasmid was identified by PCR,restriction enzyme analysis and sequencing. The homology analysis revealed that the nucleotide sequences similar of the 3-1E gene of the E.acervulina Baodong strain with Reference sequence were 99.6%.Its highly conservative.The plasmid PGM-3-1E as a template, amplify 3-1E gene reading frame.The gene sequence of E.acervulina merozoite surface antigen 3-1E was cloned into pET28a(+)vector, constructed recombinant plasmid pET28a-3-1E, then transformed into E.coil BL21 strain to Expression. The 3-1E fusion protein band of about 22kDa was identificated by SDS-PAGE. Western blot analysis indicated that the recombinant protein could react specifically with Eimeria acervulina polyclonal antibody. The gene could synthesis a fusion protein at high levels.The DNA vaccine pcDNA3-3-1E was constructed by inserting ORF sequence of 3-1E into eukaryotic vector pcDNA3.l(+). The plasmid was identified to be positive plasmid by enzyme cleavage and PCR. Sequencing analysis indicated the gene insert eukaryotic vctor pcDNA3-3-1E is correct.After purification of the recombinant plasmid inoculate a-week-chickens as DNA vaccine.After the purification of recombinant expression plasmid pcDNA-3-3-1E 50μg injection of 7-day-old chicks, one week after the booster immunization, 21 days old when taking the injection site and non-injection site muscle tissue, using RT-PCR used to detect target gene.The results showed that the dose of 50μg test chickens can produce stronger resistance effect:Insects attack the egg sac of chicken production fell 67.67 percent, lower intestinal lesion score 66.96%, and the relative weight of chicken rate of 88.36%, ACI value of 167.06.The plasmid vaccine pcDNA3-3-1E after purification was injected into one-week-old chicken leg i.m(50μg per bird).The transcription of the recombinant gene was identified through RT-PCR at the 7th day post immunization. The results showed that the transcripted products of the plasmid were detectable in the injected site. Nothing was detected in the uninfected site. The conclusions were that the DNA vaccines were successfully constructed and able to express in vivo.