丝状支原体山羊亚种特异性蛋白基因Mmc-3740的克隆表达和免疫原性分析

张靖鹏; 江锦秀; 林裕胜; 游伟; 胡奇林

doi:10.19303/j.issn.1008-0384.2019.10.002

丝状支原体山羊亚种特异性蛋白基因Mmc-3740的克隆表达和免疫原性分析

doi: 10.19303/j.issn.1008-0384.2019.10.002

福建省农业科学院畜牧兽医研究所，福建　福州　350013

基金项目: 国家重点研发计划项目（2016YFD0500906）；福建省农业科学院科技创新团队建设项目（2017-09）；福建省科技计划公益类专项（2019R1026-17）；福建省农业科学院青年人才创新基金（YC-20180-3）福建省农业科学院科技创新团队建设项目（PC2018-8）

详细信息

作者简介:
张靖鹏（1988−），男，硕士，主要从事动物传染病研究（E-mail: 870063543@qq.com）

通讯作者:
胡奇林（1963−），男，硕士，研究员，主要从事动物传染病学和免疫学研究（E-mail: hql562713@163.com）

中图分类号: S 852.62
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出版历程
- 收稿日期: 2019-07-15
- 修回日期: 2019-09-01
- 刊出日期: 2019-10-01

Cloning and Expression of Specific Protein Gene, Mmc-3740, of Mycoplasma mycoides

Institute of Animal Husbandry & Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350013, China

摘要

摘要: 目的构建表达丝状支原体山羊亚种特异性蛋白基因Mmc-3740的重组表达菌，为Mmc-3740蛋白的进一步研究与应用奠定基础。方法采用PCR技术从Mmc-47中扩增出该基因，进行克隆、测序，将克隆质粒与pET-28a分别双酶切，回收正确的片段进行连接构建重组表达质粒，转化至表达菌BL21(DE3)中，IPTG诱导表达，产物用SDS-PAGE 检测，用镍柱对其纯化后免疫小鼠获得高免血清，并进行Western-blot验证。结果成功构建了克隆和表达载体，重组菌表达目的蛋白大小约21 ku。表达产物通过镍柱纯化后可得到单一的目的蛋白条带，Western-blot检测结果显示所表达的目的蛋白有较好的免疫原性。结论成功在BL21(DE3)宿主菌内表达了Mmc-3740重组蛋白。
- 丝状支原体山羊亚种 /
- 特异基因 /
- 原核表达 /
- 免疫原性
Abstract: Objective To construct a recombinant bacterium that expresses the specific gene Mmc-3740 of Mycoplasma mycoides subsp. capri for further study and application of the protein. Method Mmc-47 was amplified by PCR, then cloned and sequenced. The cloned plasmids and pET-28a were undergone a double digestion to recover the correct fragments for ligation and construction of plasmid with the recombinant expression. Verified by sequencing, the plasmid was transformed into the expression strain, BL21 (DE3). The expression was induced using IPTG to obtain the products to be identified by SDS-PAGE and purified through the nickel affinity chromatography to produce hyperimmune serum against mouse. The serum was then identified by the western-blot for confirmation. Results The cloning and expression vectors were successfully established. The molecular weight of the recombinant protein was approximately 21 ku. The expressed product was purified showing an apparent immunogenicity. Conclusion The Mmc-3740 recombinant protein was successfully expressed in BL21 (DE3) to provide the basis for further study on the functions and characteristics of the gene.
- Mycoplasma mycoides subsp.capri /
- specific genes /
- prokaryotic expression /
- immunogenicity

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图 1 目的片段的PCR扩增

注：1. DNA 标准 DL2000；2. 目的基因片段；3. 阴性对照。

Figure 1. PCR amplification of target fragment

Note: 1. DNA maker DL2000; 2. product of target gene fragments; 3. negative control.

下载: 全尺寸图片幻灯片

图 2 重组质粒的双酶切

注：1. DNA 标准 DL5000；2. 重组质粒双酶切产物；3. 阴性对照。

Figure 2. Double-enzyme digestion of recombinant plasmids

Note: 1. DNA maker DL2000; 2. products of recombinant plasmid digestion; 3. negative control.

下载: 全尺寸图片幻灯片

图 3 诱导表达的结果

注：1. 蛋白分子量标准；2. 空载体；3. 诱导的pET-28a-3740。

Figure 3. Results of induced expression

Note: 1. protein molecular weight marker; 2. blank vector; 3. expression vector pET-28a-3740.

下载: 全尺寸图片幻灯片

图 4 重组蛋白Western bolt结果

注：1.蛋白分子量标准；2. 重组蛋白与其高免血清的免疫印迹；3.阴性对照。

Figure 4. Immunoblotting of recombinant protein

Note：1. protein molecular weight marker; 2. immunoblotting of recombinant protein with its antiserum; 3. negative control.

下载: 全尺寸图片幻灯片

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