Eukaryotic Expression And Purification Of The Leghorn Chicken FOXL2Gene

FOXL2was considered to be the first human autosomal genes, plays an important role inmaintaining ovarian function. It also is the earliest discovered in vertebrate gender dimorphism ovarydifferentiation markers. FOXL2gene is not only plays an important role in fight agasinst the follicleapoptosis and maintaining the follicle reserve function, but also play a important role in chicken layingperformance. At present, as people of FOXL2function research gradually thorough, the demand forFOXL2protein also increased, but the market existing FOXL2products have the features of little andexpensive, not be satisfied with good application. Based on this, the lab used e. coli expression system forleghorn FOXL2expression, but has less protein and low activity. This study used yeast expression systemfor leghorn FOXL2eukaryotic gene expression and purification, in order to get a lot of expression of theprotein.This research is based on the whole genome sequences of leghorn FOXL2in the GenBank（registration number: JF₇08868.1）. By NCBI comparative analysis software, to analyze its conservativesequence, and then according to the multi-clone restriction enzyme sites of eukaryotic expression vector.pPICZaA, using Primmer5.0to design and synthesis a pair of primers（P1and P2）. Using the SPF Leghornwhole blood as the template, by PCR amplification with enzyme FOXL2gene loci, directional cloning toeukaryotic expression vector pPICZaA, build pPICZaA-FOXL2recombinant eukaryotic expression vector.The recombinant plasmid with SacI pPICZaA-FOXL2after linearization, DianZhuan yeast GS115andsummarized.28℃cultivate5d, after PCR, and add methanol The recombinant expression plasmid iwasinduced96h by methanol on the condition of28℃. Using SDS-PAGE and Western-blot to analyze theexpression of recombinant proteins and identification the antigen specificityof recombinant protein. Thepurified products of recombination proteins was obtained by His column affinity.Results showed that the target genes of FOXL2was successfuly amplified, and afteridentification by PCR, enzyme digestion and sequencing analysis, the recombinant clone vectorpMD18-T-FOXL2and the recombinant expression vector pPICZaA-FOXL2were constructed successfully.It showed that the induced expressed protein was about37ku through the SDS-PAGE analysis. And theWestern blot revealed that the expressed protein was the His tag fusion protein, which indicated that thefusion protein was expressed successfully by the recombinant eukaryotic expression vector. After a lot of induction and purification of the protein, get a lot of high concentration of purified protein. The expressionand purification of the Leghorn FOXL2gene established the foundation for further study on its biologicalfunction.