%0 Journal Article
%T 犬卵透明带3蛋白原核表达和抗血清的制备<br>The Prokaryotic Expression of Canine Zona Pellucida 3 Protein and Preparation of its Antiserum
%A 张贝贝
%A 王颖
%A 张冀
%A 张富春
%J 四川动物
%D 2016
%X 中文摘要：在研发控制流浪犬卵透明带3（CZP3）免疫不育疫苗的过程中，需要制备特异性的CZP3抗血清进行免疫检测。本研究克隆获得CZP3的cDNA（全长1 281 bp）和除去跨膜区和信号肽的核心片段（ CZP3 C，984 bp），通过构建原核表达载体pET28a- CZP3 C，转化大肠杆菌BL21（DE3）。在28℃下以0.6 mmol·L -1异丙基硫代β-D-半乳糖苷诱导表达4 h，获得CZP3 C融合蛋白。利用CZP3 C融合蛋白免疫新西兰大白兔，制备特异性的抗血清，并对抗血清的特异性和效价进行评价。SDS-PAGE结果表明，融合蛋白以包涵体形式存在。经过镍柱亲和纯化及8 mmol·L -1尿素复性后，获得了高纯度的CZP3 C融合蛋白。用0.8 μg/μL -1的CZP3 C融合蛋白免疫新西兰大白兔，免疫后的兔抗血清用Western blot及间接免疫荧光实验检测，结果证明CZP3 C兔抗血清具有较高的特异性，ELISA检测效价为1：409 600。本研究所制备的抗血清能够为流浪犬免疫不育疫苗的研制提供检测材料。<br>英文摘要：To develop the canine zona pellucida 3 (CZP3) contraceptive vaccine that could be used to control the birth rate of stray dogs, antiserum is required to carry out immune detection. In this study, the full length cDNA of CZP3 (1 281 bp) and the core fragment of CZP3 ( CZP3 C, 984 bp) without the transmembrane domain and the signal peptide were cloned, respectively. CZP3 C was ligated to pET28a vector to generate the recombinant plasmid pET28a- CZP3 C, and then the plasmid was transferred into Escherichia coli BL21(DE3). The expression of CZP3 C fusion protein was induced with 0.6 mmol·L -1 isopropyl β-D-1-thiogalactopyranoside (IPTG) at 28℃ for 4 h. The specific antiserum of CZP3 C fusion protein was obtained from the rabbit after immunization, and the specificity and titer of the prepared antiserum were evaluated. The results of SDS-PAGE showed that CZP3 C mainly existed in the form of inclusion bodies. High purity of the fusion protein was obtained after nickel column affinity purification and 8 mmol·L -1 urea renaturation. Two Newzealand rabbits were immunized with CZP3 C of 0.8 μg·μ L -1, respectively, the generated polyclonal antiserum was detected with Western blot and indirect immunofluorescence. The results showed that the anti-CZP3 C rabbit serum had high specificity, and the titer was 1:409 600 as determined by the enzyme linked immunosorbent assay (ELISA). The prepared antiserum can provide testing materials for the development of stray dog contraceptive vaccines. 2016,35(5): 697-702 收稿日期：2016-05-31 DOI：10.11984/j.issn.1000-7083.20160145 分类号：Q78;Q959.8 基金项目：新疆维吾尔自治区科技支疆项目（201591134） 作者简介：张贝贝(1989-),男,硕士研究生,研究方向:分子免疫学,E-mail:1056203290@qq.com *通讯作者：张富春,博士生导师,研究方向:分子与生物化学,E-mail:zfcxju@qq.com 参考文献： 范翠英, 冯利兴, 樊金玲, 等. 2012. 重组蛋白表达系统的研究进展[J]. 生物技术, 22(2): 76-80. 王毅飞, 蔡德鸿, 陈宏, 等. 2009. His-GILI原核表达载体的构建及其表达与纯化[J]. 广东医学, 30(2): 191-193. 杨川, 胡敏. 2016. 斑马鱼 SFPQ 蛋白的原核表达及纯化[J]. 生物技术通报, (1): 163-168. Chen DY, Chen YM, Tsai WC, et al.2015. Significant associations of antidrug antibody levels with serum drug trough levels and therapeutic response of adalimumab and etanercept
%K 犬
%K 卵透明带3
%K 原核表达
%K 融合蛋白
%K 抗血清&lt
%K br&gt
%K 英文关键字：dog
%K zona pellucida 3
%K prokaryotic expression
%K fusion protein
%K antiserum
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