【摘要】：根據(jù)雞的蛋白激酶R樣內(nèi)質(zhì)網(wǎng)激酶(PERK)基因序列進(jìn)行抗原肽分析,選擇含大片段抗原表位區(qū)約1 500 bp的一段基因(15~1 515 bp)設(shè)計(jì)一對(duì)特異性引物后進(jìn)行RT-PCR擴(kuò)增,并構(gòu)建pET-32a(+)-PERK重組質(zhì)粒,將其轉(zhuǎn)化至BL21(DE3)感受態(tài)細(xì)胞中.優(yōu)化誘導(dǎo)表達(dá)條件,純化重組蛋白后免疫兔并制備多克隆抗體.PCR鑒定、酶切鑒定和測(cè)序結(jié)果表明,pET-32a(+)-PERK重組質(zhì)粒構(gòu)建成功.SDS-PAGE電泳鑒定結(jié)果表明,重組蛋白在1.0 mmol·L-1IPTG、25℃下誘導(dǎo)9 h時(shí)的表達(dá)量最大.蛋白純化結(jié)果表明,100 mmol·L-1咪唑洗脫液可較好地洗脫重組蛋白,獲得較多的純化蛋白.免疫結(jié)束后,抗體效價(jià)檢測(cè)結(jié)果表明,制備的多克隆抗體效價(jià)達(dá)1∶32 000,可以與重組蛋白特異結(jié)合.
[Abstract]:According to the sequence of protein kinase R-like endoplasmic reticulum kinase (PERK) gene, a pair of specific primers (15515bp) were designed and amplified by RT-PCR. The recombinant plasmid of pET-32a () -Perk was constructed. It was transformed into BL21 (DE3) competent cells. After the recombinant protein was purified, the rabbit was immunized with polyclonal antibody .PCR. The results of restriction enzyme digestion and sequencing showed that the recombinant plasmid pET-32a () -Perk was successfully constructed and identified by SDS-PAGE electrophoresis. The expression of recombinant protein was the highest at 1.0 mmol L ~ (-1) IPT GG at 25 鈩，

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