【摘要】：桑樹皺葉病毒(mulberry crinkle leaf virus,MCLV)是近年從感病桑樹中分離鑒定的一種新的桑樹病毒,該病毒屬于雙生病毒科。由于MCLV外殼蛋白基因(cp)序列中含有較多大腸埃希菌(Escherichia mori)BL21(DE3)的稀有密碼子而限制了其在大腸埃希菌中的表達(dá)。通過人工合成DNA的方法對(duì)MCLV cp基因序列進(jìn)行優(yōu)化,將其中40個(gè)大腸埃希菌稀有密碼子變換成同義偏愛密碼子,構(gòu)建優(yōu)化后的MCLV cp基因的原核表達(dá)載體p GEX4T-MCLV CP并轉(zhuǎn)化大腸埃希菌BL21(DE3)菌株,經(jīng)0.1 mmol/L IPTG誘導(dǎo)及SDS-PAGE電泳鑒定,實(shí)現(xiàn)了融合蛋白GST-MCLV CP在大腸埃希菌BL21(DE3)中高效表達(dá)。通過SDS-PAGE純化后切膠回收原核表達(dá)的融合蛋白GST-MCLV CP,以其為抗原免疫新西蘭大白兔制備MCLV CP的多克隆抗體,經(jīng)間接ELISA法測(cè)定該抗血清的效價(jià)為1∶4 096,Western blot檢測(cè)其能與MCLV發(fā)生特異性反應(yīng)。用制備的抗血清對(duì)MCLV感染陽(yáng)性的桑葉樣品進(jìn)行間接ELISA檢測(cè),其檢出率僅為25%,但將桑葉樣品的粗汁液煮沸處理后能顯著提高M(jìn)CLV的檢出率,檢出率達(dá)83.3%。
[Abstract]:Mulberry wrinkled leaf virus (mulberry crinkle leaf virus,MCLV) is a new mulberry virus isolated and identified from susceptible mulberry in recent years, which belongs to Geminiviridae. The expression of MCLV coat protein gene (cp) contains more rare codon of Escherichia coli (Escherichia mori) BL21 (DE3), which limits its expression in Escherichia coli. The sequence of MCLV cp gene was optimized by synthetic DNA, and 40 rare codon of Escherichia coli were transformed into synonymous preference codon. The optimized prokaryotic expression vector p GEX4T-MCLV CP of MCLV cp gene was constructed and transformed into Escherichia coli BL21 (DE3) strain. It was induced by 0.1 mmol/L IPTG and identified by SDS-PAGE electrophoresis. The fusion protein GST-MCLV CP was highly expressed in Escherichia coli BL21 (DE3). The polyclonal antibody of MCLV CP was prepared by immunizing New Zealand white rabbits with the fusion protein GST-MCLV CP, which was purified and purified by SDS-PAGE, and the titer of the antiserum was determined to be 1:4 096 by indirect ELISA assay. Western blot detected that it could react specifically with MCLV. The indirect ELISA detection of MCLV positive mulberry leaf samples was carried out with the prepared antiserum, and the detection rate was only 25%. However, the detection rate of MCLV was significantly increased by boiling the crude juice of mulberry leaf samples, and the detection rate was 83. 3%.
【作者單位】： 江蘇科技大學(xué)生物技術(shù)學(xué)院;農(nóng)業(yè)部蠶桑遺傳改良重點(diǎn)實(shí)驗(yàn)室中國(guó)農(nóng)業(yè)科學(xué)院蠶業(yè)研究所;
【基金】：江蘇省自然科學(xué)基金項(xiàng)目(No.BK20151320)
【分類號(hào)】：S888.71
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本文編號(hào)：2492724