Cloning Of TA4 And EtMIC₄ From The Eimeria Tenella And Construction Of Their Prokaryotic Expression Vectors

The TA4 gene and EtMIC₄ gene were cloned and sequenced, and its prokaryotic expression vectors were constructed to gain the genenal information for further studying recombinant vaccine against coccidiosis in this paper. According to the reporded sequences, two pair of primers were designed and synthesized, two genes from sporozoited oocysts of E.tenella (shanghai strain) were amplified by RT-PCR. The PCR products were checked by agrose gel electrophoresis and purified by agrose gel fraction method.The purified products of TA4 were ligated to pGEM-T-easy vector successfully. The ligated products were transferred into content cell DH5a. The specific recombinant plasmid was identified by PCR and restriction endonuclease analysis. The results indicated that the resultant construct contained the gene of interest TA4 at right orientation of the insert. The positive cloned was sequenced by Sanger's dideoxy sequencing method. The results demonstrated the size of TA4 gene was 773 bp in length and included an open reading frame encoding a protein of 216 amino acid residues. The cDNA of TA4 (SH) was identical with TA4(M21004.1) and shared a high of 99% identity with that of reported TA4(ZJ)(DQ327836.1). and at the 38 bp,51 bp,229 bp,462 bp and 635 bp of the sequence of TA4 gene, the bases mutated respectively from G,A,G,T,T into A,C,A,A,C, but the mutation did not rusult in change of open reading frame. The reasons given rise to reduce nucleotides may be there was a genetic diverse in different Eimeria strains. Furthermore, the fragment encoding the TA4 gene was excised from the positive clone pGEM-TA4 by EcoR I and Hind III and purified by agrose gel fraction method. Then the fragment was subcloned into the pET-28-c expression vector digested by EcoR I and Hind HI restriction enzymes; The fragment encoding the TA4 gene was excised from the positive clone pGEM-TA4 by EcoR I and Sal I and purified by agrose gel fraction method. Then the fragment was subcloned into the pGEX-4T-2 expression vector digested by EcoR I and Sal I restriction enzymes.The recombinant expression plasmids were identified by PCR and restriction enzymes analysis. The results indicated the fragment was correctly inserted into the pET-28-c and pGEX-4T-2 expression vector and conformed to a reading frame. The fusion protein were expressed in E.coli BL21 (DE3 ) host with a predicted molecular weight of 25 kDa and 53 kDa. The purified products of EtMIC₄ were digested by EcoR I and Hind HI, Then the fragments were ligated into the pET-28-a and pET-32-a expression vectors digested by EcoR I and Hind III restriction enzymes. The ligated products were transferred into content cell DH5a. The specific recombinant plasmid was identified by PCR and restriction endonuclease analysis. The positive cloned was sequenced by Sanger ' s dideoxy sequencing method. The results demonstrated the size of EtMIC₄ gene was 1351 bp in length and included an open reading frame encoding a protein of 315 amino acid residues.. Compared with the EtMIC₄, EtMIC₄(SH) was inserted 125 bases at the 5510 bp of EtMIC₄.The reasons given rise to reduce nucleotides may be there was a genetic diverse in different Eimeria strains.