Profiling The Cloning And Expressing Of Chicken Lymphotactin Gene

The ORF sequence of the gene encoding the chicken lymphotactin (ChLptn) was cloned by RT-PCR using the specific primers designed according to the cDNA sequence by Rossi et al(1999). The total RNA was extracted from the isolated lymphocytes of Coban chicken spleen and used as the template for RT-PCR. The RT-PCR product was cloned into the pGEM-T-easy plasmid vector when purified by agarose gel electrophoresis.The ligated product was transformed into E.coli DH 5 α , and the positive transfomants identified by PCR were transferred into LB medium for extracting its plamid for further identification by EcoR I digestion. The cloned ORF region was sequenced and the result showed the nucleotide sequence of the cloned chLptn gene was 99.66% identifical to the ChLptn gene sequence listed in GenBank.The cloned ORF was 294 bp in full sequence with an coding region arranged from its 1^th to 291^th bp, encoding a peptide with 97 amino acids. The encoding protein predicted with DNAssist V1.02 is 11kDa in molecular weight with pI 10.9.The ORF was amplified by PCR using specific primers designed according to the cloned sequence, and their ligated products with the plasmid vector pET-32a(+) was transformed into E.coli BL21 (DE3) by CaCl₂ method. The transformants were identified by PCR amplification and endonuclease digestion.The sequence of positive clones wasanalyzed, and was expressed under the induction of lmM IPTG The SDS-PAGE assay showed that the molecular weight of recombinant ChLptn was 31kDa. The recombinant ChLptn was expressed in a large amount at 1 hour after IPTG induction and maximally expressed at 4 hours .The ORF sequence with and without signal peptide sequence were amplified using specific primers designed according to the cloned ORF sequence, and ligated with the plasmid vector pPIC9k. The ligated products were transformed into E.coli DH 5 a by CaCh method. After the identification of endonuclease digestion, the sub-sequences from the positive transformants were recombinant with Pichia strains GS115.Then the recombinants were cultured on MD and MM plates for screening the HisMut+ colonies.The result revealed by the PCR detecting the transformants better growing on MD but not or poorly on MM plates showed that we constracted the recombinant Pichia yeast strains successfully.