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犬卵透明带3蛋白原核表达和抗血清的制备
The Prokaryotic Expression of Canine Zona Pellucida 3 Protein and Preparation of its Antiserum
张贝贝, 王颖, 张冀, 张富春*
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作者单位:新疆大学生命科学与技术学院, 新疆生物资源基因工程重点实验室, 乌鲁木齐 830046
中文关键字:犬;卵透明带3;原核表达;融合蛋白;抗血清
英文关键字:dog; zona pellucida 3; prokaryotic expression; fusion protein; antiserum
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(CZP3C,984 bp),通过构建原核表达载体pET28a-CZP3C,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CZP3C融合蛋白。利用CZP3C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CZP3C融合蛋白。用0.8 μg/μL-1的CZP3C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CZP3C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。
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 (CZP3C, 984 bp) without the transmembrane domain and the signal peptide were cloned, respectively. CZP3C was ligated to pET28a vector to generate the recombinant plasmid pET28a-CZP3C, and then the plasmid was transferred into Escherichia coli BL21(DE3). The expression of CZP3C 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 CZP3C 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 CZP3C 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 CZP3C 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-CZP3C 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
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