Cloning, Bioinformatic Analysis And Prkaryotic Expression Of Chicken Lif And Pouv Gene

Leukaemia inhibitory factor（LIF）, a member of IL-6familiy, is a cytokine with multiple effect. It can effect on different types of cells in various ways. The most significant effect of LIF is that it can inhibit the differentiation of embryonic sten cells and maintains their proliferation. Encoded by POU5f1gene and expressed in the undifferentiated embryonic stem cells（ES cells）, transcription factor Oct4is not only a member of the subfamily V of POU family, but also a key factor for reprogrammring somatic cells to the induced pluripotent stem cells. The differentiation direction of ES cells depends on expression level of Oct4in these cells. The purpose of this paper is to clone chicken LIF and POUV, to analize their characteristics bioinformatically, finally to construct their prokaryotic expression vector and further to induce expression.Experiment1. Extracted from an adult chicken liver tissue, the total RNA was used as a templateto amplify chicken LIF gene by by RT-PCR technigues, and the resulting PCR product was cloned to pEASY-T1cloning vector to get a recombinant plasmid pEASY-T1-chLIF. Removed from a DNA sequencing confirmed plasmid pEASY-T1-chLIF, the chicken LIF cDNA was subcloned to pET-30a to construct the prokaryotic expression vector pET-30a-chLIF. The results showed that:（1）Recombinant plasmid pEASY-T1-chLIF and pET-30a-chLIF were constructed. The cloned chiken LIF consists of564bp nucleotide acids, encoding188amino acids, which respectively share99.8%and99.5%homology with another Gallus LIF homolog （XM₄18264） on nucleotide and amino acid sequences, without mutation of insertion or deletion.（2） Multiple alighment of LIF homologes among different spices revealed that chicken LIF shares lower conservation with those of mammalian, indicating that LIF is of speciesspecificific.（3） Theoreticlally, the molecular weight of chicken LIF protein is20.885KDa, and its isoelectric point is9.342.（4） chicken LIF protein is hydrophilic protein without transmembrane domains.（5） The secondary structure of chicken LIF protein is composed of48.66%random coil,43.32%alpha helix and8.02%extended strand. Tertiary structure is composed of four helixs which connected by rings.Test2.The experimental procedures were the some as above. The difference was that the total RNA was extracted from the testis of new born rooster. pMD18-T was used as cloning vector. The recombinant plasmid pET-30a-chPOUV was transformed into E.coli BL21（DE3） and expressed by the induction of IPTG. The fusion protein was identified with SDS-PAGE analysis and Western blotting. The result show that:（1） Recombinant plasmids pMD18-T-chPOUV and pET-30a-chPOUV were respectively constructed, and the cloned chicken POUV gene consists of888bp nucleotide acids, encoding295amino acids, which respectively share99.4%and98.6%homology with another Gallus POUV homolog （NM₀01110178） on nucleotide and amino acid, without mutation of insertion or deletion.（2） Multiple alighment of POUV homologes among different spices revealed that, in spite of its species-specificificity, chicken POUV shares higher conservation with a domain of human POUV polypeptide, which located from the89th to227th amino acids, indicating it may be the function domain of chicken POUV protein.（3） Theoreticlally, the molecular weight of chicken POUV protein is33.229KDa, and its isoelectric point is9.791.（4） Chicken POUV protein is a hydrophilic protein without transmembrane domains. There is no signal peptide at N terminus of chicken POUV protein.（5） The secondary structure of chicken POUV protein is composed of53.22%random coil,33.56%alpha helix and13.22%extended strand.Tertiary structure is composed of two multi-helix structures which connected by a ring.（6） SDS-PAGE analysis and Western blotting showed that the recombinant plasmid pET30a-chPOUV was expressed in E.coli BL21（DE3）, the molecular weight of His-chPOUV fusion protein is about41kDa.In a word, chicken LIF and POUV genes were cloned, and their prokaryotic expression vectors were also constructed, than chicken POUV was expressed in prokaryotic. This paper will lead to get purified LIF protein, and prepare polyclonal or monoclonal anti-POUV antibody, which could be used in culture of chicken ES cells and in derivation of chicken iPS cells.