Cloning And Sequences Analysis Of Guangxi Swamp Type Buffalo Cytokines Gene

In this work, the total RNA was extracted from peripheral blood mononuclear cells (PBMC), isolated from healthy swamp type buffalo and stimulated with ConA. And the cDNAs of buffalo IL-2, IL-4, IL-5, IL-10, IL-12(P35, P40), IL-18 and TNF-αgene was amplified by reverse transcription polymerase chain reaction(RT-PCR). The PCR products were cloned into pMD18-T vector. Then, the positive recombinant clones were sequenced and analyzed.Sequence analysis showed that the 724 bp IL-2 cDNA fragment contains ORF(468bp), encoding 155 amino acid and having a molecular weight of approximate 17.5kD. The nucleotide and the deduced amino acide sequences of the buffalo IL-2(Guangxi) share 65.2%-99.6% and 49%-100% homology with that of buffalo(India), bovine, sheep, goat , human and mouse, respectively.IL-4 gene cloned from PBMCs shares 98.8%, 99.3%, 94.1%, 84.8%, 80.6% nucleotide homology with that of buffalo(India), bovine, sheep, pig and horse, respectively; and the deduced amino acide sequence shares 96.3%, 98.5%, 90.4%, 78.4% and 59.6% homology with that of buffalo(India), bovine, sheep, pig and horse, respectively. Its full length is 469bp, containing 408bp ORF, encoding 135 amino acid and having a molecular weight of approximate 15.0kD.IL-5 gene cloned from PBMCs has length of 430bp, containing ORF(405bp), encoding 134 amino acid and having a molecular weight of approximate 15.3kD. The cDNA shares 99.0%, 96.5%, 89.6% and 89.1% nucleotide homology with that of bovine, sheep, pig and horse, respectively; and the deduced amino acide sequence shares 97.8%, 96.2%, 85.9% and 86.7% homology with that of bovine, sheep, pig and horse, respectively. The 536bp IL-10 cDNA contain ORF, encoding 178 amino acid and having a molecular weight of approximate 22.4kD. The nucleotide and the deduced amino acide sequences of the buffalo IL-10(Guangxi) share 75.9%-99.6% and 74.2%-100% homology with that of buffalo(India), bovine,sheep,pig,horse,human and mouse, respectively.Buffalo IL-12(p35 , p40) gene have been cloned successfully with-full length of 694bp(ORF 666bp)and 1013bp(ORF 984bp), respectively. Homology of nucleotide between the cloning sequences and the suquences of buffalo IL-12 p35 and p40 cDNA registered in the GeneBank are 99.5% and 99.4%. Homology of amino acid sequence are 98.6% and 98.5%. p35 encodes 222 amino acid and having a molecular weight of approximate 24.86kD. p40 encodes 328 amino acid and having a molecular weight of approximate 37.03kD.The 602bp IL-18 cDNA contains ORF(582bp), encoding 193 amino acid and having a molecular weight of approximate 22.18kD. The nucleotide and the deduced amino acide sequences of the buffalo IL-18(Guangxi) shares 67.7%-99.3% and 60.6%-98.5% homology with that of buffalo(India), bovine, sheep, goat, pig, horse, human and mouse, respectively.Buffalo TNF-αgene has been cloned successfully with full length of 715bp(ORF 705bp). It encodes 234 amino acid and having a molecular weight of approximate 25.56kD. The nucleotide and the deduced amino acide sequences of the buffalo TNF-α(Guangxi) share 73.0%-99.7% and 70.6%-98.7% homology with that of buffalo(India), bovine, sheep, goat, pig and human.In this work, we have cloned buffalo IL-2, IL-4, IL-5, IL-10, IL-12 (p35,p40) , IL-18 and TNF-αgene successfully. This provides a good foundation for further research on buffalo cytokines biological characteristics and application. The availability of these cytokine molecules will aid in the study of their role in the immunology and pathogenesis of infections in water buffalo.