Prokaryotic Expression And Construction Of Eukaryocyte Expression Plasmid Of Eimeria Tenella 3-1E Gene And Its Immunoprotective To Chicken

Coccidiosis in poultry is caused by protozoan parasites of the genus Eimeria. There are 9 species of chicken coccidia and the most virulent specie is Eimeria tenella (E. tenella). The disease was difficult to control with worldwide distribution.The current means of prevention and control coccidiosis depends on the drug, but there are some problems such as the drug resistance, the high cost of developing new drugs, drug residues and environmental protection. A better means of prevention and control are eager to be found to replace drug treatment.The Eimeria 3-1E gene as a protective gene is high conserved between different poultry Eimeria sp.. The target gene was cloned into the prokaryotic expression vector pET-32a(+) and the eukaryotic expression vector pcDNA3.1(+). The chicken IFN-γgene and chicken IL-2 gene were inserted into the 3-1E gene downstream in eukaryotic expression vector respectively. Because chicken IFN-γand chicken IL-2 play an important role in the anticoccidial immune. In order to observe the induced specific immune response, the recombinant antigen and eukaryotic recombinant DNA plasmids were immunized animals to gain the protective effect. The results showed:1.The constructed prokaryotic expression plasmid pET-32a(+)-3-1E was digested by restriction enzyme and there were two obvious bands of 6000bp and 500bp. Sequence determination showed the correct sequence. It is suggested that pET-32a(+)-3-1E plasmid was successfully constructed.2.The plasmid pET-32a(+)-3-1E was transformed into Rosetta competent cells and carried out the prokaryotic expression. The 3-1E antigen was expressed as a soluble fusion protein. Then the optimized inducing time and concentration of IPTG was selected and the 6h and 0.8mmol/L were the best expression conditions. The western-blot test proved that the recombinant protein can combine with sera of chickens infected Eimeria tenella.3.The immunization protection test of recombinant 3-1E protein was carried out. There were 5 groups:recombinant protein group, recombinant protein + Freund's complete adjuvant (FCA) control group, drug control group, red control group, white control group. The results showed that recombinant 3-1E protein can enhance the different levels of serum IgG, IL-4, IFN-γ, but there was little proliferation function on the splenic T lymphocyte. And there was a small rate of increase after all the indexs in the recombinant protein + Freund's complete adjuvant (FCA) control group. The anticoccidial index (ACI) of recombinant protein group was 171.45. And it was 174.92 of the recombinant protein + Freund's complete adjuvant (FCA) control group. Both of them are with moderate anticoccidial effect.4.The E.tenella 3-1E gene; chicken IFN-y gene and the chicken IL-2 genes were tandemed in eukaryotic expression vector pcDNA3.1(+) to construct eukaryotic expression plasmid pcDNA3.1(+)-3-1E-IFN-γ, pcDNA3.1(+)-3-1E-IL-2. The control plasmid pcDNA3.1(+)-3-1E, pcDNA3.1(+)-IFN-γand pcDNA3.1(+)-IL-2 were also constructed. The results were identical with the theory(pcDNA3.1(+)vector 5428bp,3-1E gene 513bp, chicken IFN-y gene 495bp and chicken IL-2 gene 429bp) after restriction enzyme digestion, PCR identification and sequencing. It was proved that these plasmids were successfully constructed.5.The plasmid pcDNA3.1(+)-3-1E-IFN-γand pcDNA3.1(+)-3-1E-IL-2 were separately inserted into chickens to detecte the gene transcription and expression in vivo. The results showed that the plasmid pcDNA3.1(+)-3-1E-IFN-y and pcDNA3.1(+)-3-lE-IL-2 could work normally in vivo.6. The chicken were immunized by the eukaryotic expression plasmids. The results showed that pcDNA3.1(+)-3-1E-IFN-γand pcDNA3.1(+)-3-1E-IL-2 could significantly improve serum IgG, IL-4, IFN-γlevels, and have a greater enhancement on T lymphocyte proliferation in the spleen. It was proved the plasmid pcDNA3.1 (+)-3-1E-IL-2 was the best. The anticoccidial index (ACI) of pcDNA3.1 (+)-3-1E-IFN-γwas 181.04. The anticoccidial. index (ACI) of pcDNA3.1 (+)-3-1E-IL-2 was:187.30. Both of them were effective for anti-coccidia effect.The prokaryotic expression and eukaryotic expression plasmid were constructed by the Eimeria tenella 3-1E gene as a protective gene. The results showed that the recombinant protein was moderate anticoccidial effect and eukaryotic expression vector pcDNA3.1 (+)-3-1E-IFN-γand pcDNA3.1 (+)-3-1E-IL-2 as a DNA vaccine had high anticoccidial effect.