甜菜夜蛾卵黄原蛋白多克隆抗体制备及其在不同发育时期蛋白表达

doi:10.3864/j.issn.0578-1752.2017.22.008

摘要/Abstract

摘要： 【目的】制备甜菜夜蛾（Spodoptera exigua）卵黄原蛋白（vitellogenin，Vg）多克隆抗体，并检测甜菜夜蛾不同发育时期血淋巴中的Vg蛋白表达量变化，为进一步研究Vg转运与利用机制以及生物学功能打下基础。【方法】以羽化24 h的甜菜夜蛾雌成虫cDNA为模板，通过PCR扩增得到甜菜夜蛾Vg基因片段，其包含vitellogenin-N结构功能区。将Vg目的片段连入pMD-19T载体后进行测序，利用DNAMAN软件分析该基因序列的准确性及编码蛋白的特性。将测序正确的Vg基因片段通过Nde I和Xba I限制性内切酶连接到原核表达载体pCzn1。将重组表达载体pCzn1-Vg转入大肠杆菌ArcticExpress，离心收集诱导表达的菌液，超声波破碎菌体沉淀，取上清液与沉淀进行SDS-PAGE检测，分析Vg重组蛋白的可溶性。在不同温度、不同浓度IPTG条件下诱导表达Vg重组蛋白，筛选最优表达条件。经Ni-NTA琼脂糖凝胶亲和层析柱纯化得到了Vg重组蛋白，将该蛋白免疫新西兰白兔，制备兔抗Vg血清抗体。采用间接ELISA方法检测血清抗体的效价，并通过Western blot法检测甜菜夜蛾雌虫不同发育阶段血淋巴中Vg蛋白的表达差异。【结果】扩增所得Vg基因片段为2 091 bp，编码697个氨基酸, 预测蛋白分子量为80.88 kD。通过大肠杆菌表达出的Vg重组蛋白相对分子量为80 kD，其大小与预期的Vg重组蛋白带大小相符合。其主要以包涵体形式表达，而在上清中表达量不明显。不同温度、不同浓度IPTG条件下诱导表达Vg重组蛋白的结果显示温度为25℃，IPTG浓度为0.6 mmol·L^-1时，Vg重组蛋白表达量最高。继续提高温度和IPTG浓度，对提高Vg重组蛋白的表达量无明显作用，且杂蛋白增多。新西兰白兔经4次免疫后，间接ELISA法检测表明，制备的兔抗Vg抗体具有较好的灵敏度，效价达到 1﹕512 000。Western blot检测Vg蛋白在甜菜夜蛾不同发育阶段血淋巴中的表达，其杂交出的单一条带在180 kD左右。Vg蛋白在甜菜夜蛾雌蛹末期开始微弱表达。雌成虫羽化后血淋巴中Vg蛋白表达量先升高后降低，呈动态变化，到羽化48 h表达量最高，随后降低。【结论】纯化获得了Vg 重组蛋白，并明确了最优表达条件（温度为25℃，IPTG浓度为0.6 mmol·L^-1）；制备了高效价的甜菜夜蛾Vg多克隆抗体，明确了甜菜夜蛾Vg蛋白的表达规律。

关键词: 甜菜夜蛾, 卵黄原蛋白基因, 原核表达, 抗体制备, 表达模式

Abstract: 【Objective】 The objective of this study is to prepare the polyclonal antibody of Spodoptera exigua vitellogenin (Vg), investigate the expression pattern of Vg protein in female hemolymph of S. exigua at different developmental stages, and to provide a basis for studying the function and mechanism of synthesis, transportation and utilization.【Method】The fragment of Vg was amplified from the cDNA of 24-h-old female adults of S. exigua by PCR which included the vitellogenin-N region. The fragment of Vg was then inserted into the pMD-19T for sequencing. The nucleic acid sequence and the amino acids encoded by this gene fragment were analyzed by DNAMAN software. The sequenced Vg fragment was inserted into the expression vector pCzn1 by Nde I and Xba I digestion. The recombinant vector pCzn1-Vg was then inserted into Escherichia coli ArcticExpress. The E. coli ArcticExpress expressing the Vg recombinant 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 recombinant protein. The recombinant Vg protein was expressed at different temperatures and concentrations of IPTG and the optimized expression condition was achieved. The recombinant protein was purified by Ni-NTA agarose. The purified recombinant protein was used to produce polyclonal antibody via immunizing rabbit. The titer of rabbit anti-Vg antiserum was evaluated by indirect ELISA. The content of Vg in female hemolymph of S. exigua at different developmental stages was detected by Western blot. 【Result】 The length of the fragment of Vg is 2 091 bp, encoding 697 amino acids. The predicted molecular weight of Vg recombinant protein is 80.88 kD. The molecular weight of Vg recombinant protein expressed in E. coli is 80 kD, which is consistent with the predicted molecular weight. It was mainly expressed in inclusion body rather than the supernatant. The results of Vg recombinant protein content expressed at different temperatures and concentrations of IPTG showed that it was highly expressed at inducing temperature of 25℃ with 0.6 mmol·L^-1 IPTG. It had no obvious effect on boosting the Vg recombinant protein level and other protein content increased by raising temperature and the concentration of IPTG. After immunizing New Zealand white rabbits with four times, the ELISA assay showed that the rabbit anti-Vg antiserum had a good sensitivity with the titer 1﹕512 000. The Vg content in female hemolymph of S. exigua at different developmental stages was detected by Western blot. A single Vg band of approximately 180 kD was detected. Vg was first expressed at late stage of female pupa and showed a low expression level. After female adult eclosion, Vg expression was in a dynamic balance which peaked in 48-h-old female adults, then decreased. 【Conclusion】 The Vg recombinant protein was successfully purified and the optimized expression condition (temperature of 25℃ with 0.6 mmol·L^-1 IPTG) is clearly defined. The polyclonal antibody of Vg protein with high titer was obtained and the expression pattern of Vg in S. exigua is explicit.

Key words: Spodoptera exigua, vitellogenin gene, prokaryotic expression, polyclonal antibody, expression pattern

赵静，孙洋，谭永安，肖留斌，姜义平，柏立新. 甜菜夜蛾卵黄原蛋白多克隆抗体制备及其在不同发育时期蛋白表达[J]. 中国农业科学, 2017, 50(22): 4316-4324.

ZHAO Jing, SUN Yang, TAN YongAn, XIAO LiuBin, JIANG YiPing, BAI LiXin. Polyclonal Antibody Preparation of Spodoptera exigua Vitellogenin and Its Protein Expression at Different Developmental Stages[J]. Scientia Agricultura Sinica, 2017, 50(22): 4316-4324.

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