【摘要】：FOXL2是第一個被認(rèn)定在維持卵巢功能方面發(fā)揮重要作用的人類常染色體基因，也是在脊椎動物中最早被發(fā)現(xiàn)的卵巢分化的性別二態(tài)性標(biāo)記。FOXL2基因不但在抗擊卵泡凋亡、維持卵巢儲備功能方面發(fā)揮著重要作用，而且其正常表達(dá)與否可能與雞產(chǎn)蛋性能的高低有關(guān)。目前，隨著人們對FOXL2功能研究的逐漸深入，F(xiàn)OXL2蛋白的需求量也不斷增大，但是市場上現(xiàn)有的FOXL2產(chǎn)品存在少而貴的特點，不能很好的滿足于應(yīng)用�；诖瞬蛔�，本實驗室曾采用大腸桿菌表達(dá)體系對來航雞FOXL2基因進(jìn)行了表達(dá)，但蛋白量較少且活性低。故本研究利用酵母菌表達(dá)系統(tǒng)對來航雞FOXL2基因進(jìn)行真核表達(dá)及純化，，以期獲得大量的高活性表達(dá)蛋白。 本研究根據(jù)GenBank上收錄的來航雞FOXL2的全基因序列（登錄號：JF＿708868.1），通過生物信息學(xué)比對分析其保守序列及真核表達(dá)載體的多克隆酶切位點，利用Primmer5.0來設(shè)計合成一對引物P1、P2。以純系SPF來航雞全血DNA為模板，經(jīng)PCR擴增帶有酶切位點的FOXL2基因，并將其與pMD18-T載體連接，獲得重組克隆質(zhì)粒pMD18-T-FOXL2。再以pMD18-T-FOXL2為模板，經(jīng)PCR擴增帶有酶切位點的FOXL2基因，定向克隆到真核表達(dá)載體pPICZaA中，構(gòu)建pPICZaA-FOXL2重組真核表達(dá)載體。用SacI將重組pPICZaA-FOXL2線性化后，電轉(zhuǎn)GS115畢赤酵母菌。28℃培養(yǎng)5d，PCR鑒定和測序正確后，進(jìn)行高拷貝子篩選，將篩選到的高拷貝轉(zhuǎn)化子加甲醇28℃誘導(dǎo)表達(dá)96h。應(yīng)用SDS-PAGE和Western blot方法分析重組蛋白的表達(dá)情況，鑒定重組蛋白的抗原特異性，然后采用His鎳柱親和層析獲得重組蛋白的純化產(chǎn)物。 研究結(jié)果表明，成功擴增出來航雞FOXL2基因，經(jīng)PCR、雙酶切和測序結(jié)果比對分析未發(fā)生氨基酸突變現(xiàn)象，成功構(gòu)建了重組克隆載體和表達(dá)載體。重組表達(dá)質(zhì)粒轉(zhuǎn)入畢赤酵母菌后經(jīng)甲醇誘導(dǎo)，由于重組蛋白以胞外分泌的形式進(jìn)行表達(dá)，取上清SDS-PAGE分析顯示，表達(dá)出與預(yù)期大小相一致的目的蛋白條帶，其分子量為37kD。Western-blot鑒定結(jié)果顯示，重組蛋白能被His標(biāo)簽單抗所識別，具有良好的反應(yīng)原性。重組蛋白經(jīng)純化后，分別得到濃度為1.2mg/mL重組目的蛋白。FOXL2基因的克隆、表達(dá)及純化為進(jìn)一步探索該基因的生物學(xué)功能奠定了基礎(chǔ)。
[Abstract]:FOXL2 is the first human autosomal gene to play an important role in maintaining ovarian function. FOXL2 gene is not only the first sex dimorphism marker of ovarian differentiation found in vertebrates. FOXL2 gene is not only in the fight against follicular apoptosis. Maintaining ovarian reserve function plays an important role, and its normal expression may be related to the egg laying performance of chickens. At present, with the deepening of the study of FOXL2 function, the demand for FOXL2 protein is also increasing, but the existing FOXL2 products in the market have few and expensive characteristics, which can not be well satisfied with the application. Because of this deficiency, we used Escherichia coli expression system to express FOXL2 gene of Leghorn chicken, but the amount of protein was less and the activity was low. In this study, the yeast expression system was used to express and purify the FOXL2 gene of Leghorn chicken in order to obtain a large number of highly active protein. According to the whole gene sequence (accession number: JF_708868.1) of Laihang chicken FOXL2 collected on GenBank, the conserved sequence and polyclonal restriction site of eukaryotic expression vector were analyzed by bioinformatics comparison. A pair of primers P _ 1, P _ 2 were designed and synthesized by Primmer5.0. The whole blood DNA of pure line SPF was used as template. The FOXL2 gene with enzyme site was amplified by PCR and ligated with pMD18-T vector to obtain the recombinant clone plasmid pMD18-T-FOXL2.. Then using pMD18-T-FOXL2 as template, FOXL2 gene with restriction site was amplified by PCR and cloned into eukaryotic expression vector pPICZaA to construct recombinant eukaryotic expression vector pPICZaA-FOXL2. The recombinant pPICZaA-FOXL2 was linearized by SacI and electrotransferred to Pichia pastoris at 28 鈩

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