槟榔植原体膜蛋白基因的克隆及其抗血清制备

Cloning and Prokaryotic Expression of Areca Phytoplasma Membrane Protein Gene and Preparation of Its Polclonal Antiserum

根据实验室测得的槟榔植原体膜蛋白基因(Ac Pmp)序列设计1对特异引物mp-FP/mp-RP,以病样槟榔总DNA为模板扩增该Ac Pmp的编码区并连接到p MD19T simple中间载体。将测序正确的阳性菌提取质粒p MD19T-Ac Pmp,经NcoⅠ和XhoⅠ双酶切后回收Ac Pmp片段,然后以正确的编码框连接到原核表达载体p ET32a(+),转化Escherichia coli BL21感受态细胞。含有p ET32a-Ac Pmp的BL21表达菌经IPTG诱导和SDS-PAGE分析表明,在30℃,0.1 mmol/L IPTG条件下诱导4 h,可产生部分可溶性的融合蛋白。利用Ni2+-NTA亲和层析柱法进行纯化并获得高纯度的可溶性融合蛋白。将纯化后的融合蛋白多次免疫家兔,取其抗血清,以融合蛋白(1∶10 000稀释)作抗原,间接ELISA法测定抗血清效价大于125 000。Western Blot杂交结果表明槟榔植原体膜蛋白抗血清能与融合蛋白特异性结合。

Total DNA was extracted from the infected Areca and used for amplifying the membrane protein gene of Areca Phytoplasma by using primers mp-FP/mp-RP based on its sequence from previous study. The membrane protein gene was finally inserted into prokaryotic expression vector pET-32a （＋） and the recombinant plasmid pET32a-AePmp was identified by Nco I/Xho I restriction enzymes and Sanger sequencing. Subsequently the recombinant plasmid was transferred into Escherichia coli BL21 competent cell, and the positive colony was induced at 30 ℃ with 0.1 mmol/L IPTG for 4 hours. The result showed that small part of soluble fused protein was expressed and then purified by Ni2＋-NTA agarose affinity chromatography, and the high purity of fused protein was finally obtained in 500 mmol/L imidazole Elution buffer. Two rabbits were immunizeed for 3 times by using purified protein and phosphate buffer control the antiserum against Phytoplasma MP was collected and indirect. Indirect enzyme-linked immunosorbent assay （ID-ELISA） indicated that the titer of antiserum was higher than 1： 125 000. Western Blot analysis further showed that the antiserum was specifically binding with the MP fused protein.