The Cloning And Expression Of Tibetan Pig IGF-1 Mature Peptide And Its Activity Analysis

Insulin-like growth factor 1(IGF-1)found from animals serum is a multifunctional growth factor,possessing regulation of animals' growth,development,and metabolism functions.IGF-1 not only promotes cellular uptake of amino acids and glucose,increases the synthesis of protein,fat and glycogen,stimulates DNA replication,cell proliferation and differentiation,but also stimulates the gonad to secrete hormones,promotes lactation and animal small intestine development.The plasma IGF-1 level is positively correlated with body weight and weight increase and IGF-1 gene has been used as a candidate gene for regulate growth traits of pig.However,there are no reports about Tibetan pig IGF-1 mature peptide which was obtained by the method of genetic engineering.In this paper,the gene of IGF-1 mature peptide was cloned and induced in E.coli BL21 and Pichia pastoris GS115 after prokaryotic and the eukaryotic expression vector were successfully constructed.It laid the foundation for its further bioactivity research and application in the animal husbandry production.1.Cloning and sequence analysis of IGF-1gene of Tibetan pig and Yanan pigA pair of primers was designed by Sus scrofa IGF-1 gene sequence from GenBank(Accession number:DQ121132.1),the total RNAs were extracted by using trizol from the livers of Tibetan pig and used as template to amplify IGF-1 gene by RT-PCR,the amplified fragments were cloned into pMD19-T vector and the recombinant plasmids pMD19T-IGF-1 were constructed and sequenced.The sequencing results indicated that the IGF-1 gene consisted of 612 nucleotides,containing a complete ORF of 462 bp encoding 153 amino acids.The IGF-1 gene sequences shared high homology with the porcine IGF-1 gene reported by Muller et al,which had an identity of 100%in Tibetan pig and had closest relative with Sus scrofa and Hubei pig,The genetic distance between 0?0.003.The IGF-1 gene sequences shared over 90%homology with the oxen,sheep,people,dogs,deer and the genetic distance between 0.068?0.075.However it had a low identity of 87.2%and 70.5%in mice and chickens.The results show that IGF-1 gene sequences are highly conservative.2.The amplification of IGF-1 mature peptide and its induced expression in E.coli.The IGF-1 mature peptide fragment was amplified with the pMD19-T-IGF-1 as a template,then the IGF-1 mature peptide was cloned into pET32a to construct a recombinant plasmid pET-32a-IGF-1 and the recombinant plasmid pET-32a-IGF-1 was transformed into E.coli BL21(DE3).Then the pET-32a-IGF-1 fusion protein was induced to express with different IPTG concentration and induction time.And the expression culture was analyzed for it's solubility and was prepared to purify pET-32a-IGF-1 fusion protein with Ni-NTA SefinoseTM Resin.Finally,the expressing culture and purified protein were identified with the method of SDS-PACE analysis and Western Blot,and which were detected the biological activity with CCK8 method.The Tibetan pig IGF-1 mature peptidegene was 315bp,Restriction enzyme mapping and sequencing showed that expression vector is constructed successfully.The pET-32a-IGF-1 fusion protein induced in E.coli BL21(DE3)and could be expressed by induction with 0.5mmol/L IPTG 18? induction 10 hours for well expression;The fusion protein expressed in an insoluble form of inclusion bodies and a high-purity fused protein was obtained with Ni-NTA agarose purification.The expressing culture and purified protein were proved to be the pET-32a-IGF-1 fusion protein with SDS-PACE and Western Blot analysis,The analysis of CCK8 showed that the recombinant protein IGF-1 can promote porcine lymphocyte proliferation.The conclusion show that IGF-1 mature peptide gene was cloned and expressed,The fusion protein has the biological activity.3.The amplification of IGF-1 mature peptide and its induced expression of IGF-1 mature peptide sequence in P.pastoris.A Pair of primers was designed by IGF-1 gene sequence from GenBank,Add his and c myc tag in downstream primers backend,and the IGF-1 mature peptide sequence was amplified with the pMD19-T-IGF-1 as a template.Then the IGF-1 mature peptide sequence was cloned into pPIC9K to construct a pPIC9K-IGF-1 plasmid.Then the recombinant plasmid pPIC9K-IGF-1 was transformed into P.pastoris GS115 by electrotransformation.Finally,the multi-copy colonies were acquired after the transformed cells was confirmed with both PCR and MM/MD plate screening methods and screened by G418.The target proteins were induced by methanol and detected by method of using Tricine-Tris-SDS-PAGE and Dot Blot.Analysis of Tricine-Tris-SDS-PAGE and Dot Blot showed that recombinant IGF-1 mature peptide protein was successfully expressed and which the molecular weight of it was about 13KDa,The analysis of CCK8 showed that the recombinant protein IGF-1 can promote porcine lymphocyte proliferation.The results show that the eukaryotic expression plasmid pPIC9K-IGF-1 is constructed successfully and expressed efficiently in pichia GS115,and the recombinant IGF-1 mature peptide protein possesses the functions of promoting porcine lymphocyte proliferation.