灰飞虱原肌球蛋白的基因克隆、原核表达及多克隆抗体制备

doi:10.3864/j.issn.0578-1752.2014.19.007

摘要/Abstract

摘要： 【目的】通过筛选灰飞虱cDNA酵母表达文库发现原肌球蛋白（tropomyosin，Tm）能与水稻黑条矮缩病毒（Rice black-streaked dwarf virus，RBSDV）P10蛋白发生互作。研究旨在克隆灰飞虱原肌球蛋白基因（Tm），将其在原核细胞中进行表达，纯化Tm蛋白，免疫大白兔并制备其多克隆抗体，为进一步分析Tm在灰飞虱与RBSDV互作过程中的作用提供抗体条件。【方法】根据与之高度同源的Tm基因信息确定灰飞虱Tm的开放阅读框（open reading frame，ORF）。提取灰飞虱总RNA，采用RT-PCR方法从灰飞虱中克隆Tm的ORF，连接至pMD-18T载体后进行测序分析，利用DNAstar软件分析该基因序列及其编码的蛋白特性。将测序验证正确的Tm通过EcoR V和BamH I限制性内切酶酶切连接至原核表达载体pET-32a (+)。重组质粒转化大肠杆菌BL21 (DE3)，经终浓度为0.4 mmol?L^-1的IPTG诱导表达4 h后检测Tm融合蛋白的表达情况。离心收集诱导表达的大肠杆菌菌液，进行超声波破碎处理，收集上清及沉淀，采用SDS-PAGE分析融合蛋白的表达。利用Ni-NTA Agarose纯化上清中的可溶性融合蛋白，经100 mmol?L^-1咪唑洗脱和0.01 mol?L^-1 PBST溶液透析后获取纯化的Tm融合蛋白，免疫大白兔，制备多克隆抗血清，并采用间接ELISA法检测抗血清效价。纯化的Tm融合蛋白及灰飞虱总蛋白经SDS-PAGE分离后，用制备的Tm多克隆抗体进行Western blotting检测，分析抗体的特异性。【结果】序列分析显示，灰飞虱Tm的ORF大小为852 bp。采用RT-PCR克隆获得此ORF并进行测序分析，结果表明扩增所得灰飞虱Tm的ORF长为852 bp，编码283个氨基酸，理论分子量大小为32.6 kD。序列比对结果发现所编码蛋白在不同物种间保守。将Tm ORF克隆入pET-32a (+)表达载体后在大肠杆菌BL21 (DE3)中进行诱导表达，表达产物经SDS-PAGE分析显示，融合蛋白大小约为55 kD，主要以可溶性蛋白形式表达，在包涵体中也有少量表达。纯化上清中的可溶性Tm融合蛋白，免疫大白兔，制备了多克隆抗体。抗体效价测定结果显示该抗体具有较好的灵敏度，效价大于1﹕409 600。采用制备的Tm多克隆抗体检测纯化的Tm融合蛋白及灰飞虱总蛋白，分别检测到1条约55 kD和约37 kD的特异性条带，表明该抗体有较强的特异性。【结论】克隆获得了灰飞虱Tm的ORF，并在大肠杆菌BL21 (DE3)中进行了诱导表达，纯化获得了Tm融合蛋白，制备了高效价的Tm多克隆抗体。

关键词: 灰飞虱, 原肌球蛋白, 克隆, 原核表达, 多克隆抗体

Abstract: 【Objective】 By screening Laodelphax striatellus Fallén (small brown planthopper, SBPH) cDNA library, it was found that the tropomyosin (Tm) could interact with RBSDV P10 protein. To provide the antibody of Tm protein for analyzing the Tm gene’s function in the process of RBSDV and SBPH interaction, the Tm was cloned and expressed in prokaryotic system. Tm protein was purified and the polyclonal antibody was obtained by immunizing the rabbit. 【Method】 The complete open reading frame (ORF) of Tm from SBPH was determined by comparing the sequence to the highly homologous Tm in other species. The total RNA was extracted and the Tm ORF was cloned by reverse transcription polymerase chain reaction (RT-PCR). Tm was then inserted into the pMD-18T for sequencing. The nucleic acid sequence and the amino acids encoded by this ORF were analyzed by DNAstar software. Tm ORF was inserted into the expression vector pET-32a (+) by EcoR V and BamH I digestion. The recombinant plasmid was transformed into E. coli BL21 (DE3) and expressed under the induction with 0.4 mmol?L^-1 IPTG for 4 h. Tm fusion protein was detected by SDS-PAGE. The E. coli BL21 (DE3) expressing the Tm fusion protein was collected and crushed by ultrasonic. The supernatant and the precipitate were collected, respectively, and SDS-PAGE was used to analyze the expression of the fusion protein. The soluble fusion protein was purified by Ni-NTA Agarose, washed with 100 mmol?L^-1 imidazole and dialyzed with 0.01 mol?L^-1 PBST. The purified fusion protein was used to produce polyclonal antibody via immunizing rabbit. The titer of rabbit anti-Tm antiserum was evaluated by indirect ELISA. To estimate the specificity of this antibody, the purified fusion Tm protein and the total proteins from SBPH were separated by SDS-PAGE and were tested by Western blotting assays using the prepared antibody.【Result】The sequence analysis results showed that the length of complete SBPH Tm ORF was 852 bp. It was amplified by RT-PCR and the sequencing result showed that the length of Tm ORF was 852 bp, encoded 283 amino acids, and its theoretical molecular weight was 32.6 kD. The blast results indicated that the encoded protein was conserved among different species. Tm was inserted into the vector pET-32a (+) and expressed in E. coli BL21 (DE3). The SDS-PAGE result showed that the Tm fusion protein of approximately 55 kD was mainly expressed as a soluble protein and there was also a small amount in inclusion body. The soluble fusion protein was purified and the polyclonal antibody was obtained by immunizing the rabbits using the purified fusion protein. The ELISA assay showed that the rabbit anti-Tm antiserum had a good sensitivity with the titer higher than 1﹕409 600. Using the polyclonal antibody to detect the purified Tm fusion protein and the total SBPH protein, specific band (55 and 37 kD, respectively) could be observed, suggesting that this antibody has strong specificity. 【Conclusion】 The ORF of SBPH Tm was cloned firstly. The Tm fusion protein was expressed in E. coli BL21 (DE3), purified successfully and used for preparing the antibody. The rabbit anti-Tm antiserum with high titer was obtained.

Key words: Laodelphax striatellus Fallén, tropomyosin, cloning, prokaryotic expression, polyclonal antibody

徐秋芳，陈晴晴，倪海平，李硕，张金凤，周益军. 灰飞虱原肌球蛋白的基因克隆、原核表达及多克隆抗体制备[J]. 中国农业科学, 2014, 47(19): 3791-3798.

XU Qiu-fang, CHEN Qing-qing, NI Hai-ping, LI Shuo, ZHANG Jin-feng, ZHOU Yi-jun. Cloning, Prokaryotic Expression and Polyclonal Antibody Preparation of Tropomyosin Gene in Laodelphax striatellus Fallén[J]. Scientia Agricultura Sinica, 2014, 47(19): 3791-3798.

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