Cloning And Sequence Analysis Of M Gene And The Construction Of Maize Expression Vector Of S₁ Gene Of Avian Infectious Bronchitis Virus

A pair of primers were designed according to the matrix(M) sequences of IBV hi Genbank.Then the full length cDNA of M gene of 8 IBV strains isolated hi China were amplified by RT-PCR. The amplified products were cloned into the pUC18-T vector and sequenced. The results showed that the nucleotide sequence length of M gene is 678bp~681bp and the deduced amino acid sequence length is 225aa~226aa. All the sequences have been accepted by Genbank. the nucleotide sequences and deduced amino acid sequences of the M gene of 8 IBV isolates in China with the published sequences of reference strains in GenBank were analyzed and compared, the results showed that the nucleotide sequences homogeneity between them is 87.1%~100%, and the deduced amino acid sequences homogeneity is 88.1%~100%. There were deletion, insertion or mutation within the IBV isolates, the frontal 150nt showed the high variation. On the basis of the phylongenetic tree of M gene, it appeared that the 8 IBV isolates hi China fall into two gene groups.The Si gene of avian infectious bronchitis virus(SAEB4 and SAIBu) were amplified by PCR with high-fidelity polyerase (pfu) and special primers with Clal and BamHI, The purified Si PCR product and pUGFPocs vector were digested by Clal and BamHI respectively, ligeat and transformed into E. coll JM109 strain. As result, positive colonies were screened on LB plate (100ug /mL amp added). The result of PCR and enzyme digestion of plasmid proved that the recombination vector (named pUSAIB4 and pUSAIBu) was obtained . The pUSAIB4 and pUSAIBn vector was digested by Kpnl and HindIII, respectively. The Ubi-Sl-Tocs fragment was taken out, inserted into the multi-cloning site of pCAMBIA1300 vector, transformed into JM109 strain finally, positive colonies were screened on LB plate (60 g /mL Kan added). The result of PCR and enzyme digestion of plasmid proved that recombinatin vector was obtained (named pCUSAIB4 and pCUSAIBu). The successful construction of pCUSAIB4 and pCUSAIB^ vector is the key foundation of Si gene expressed in maize.