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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (11): 1-8    DOI: 10.13523/j.cb.20181101
研究报告     
人源CT55蛋白原核表达及单克隆抗体的制备 *
任建委1,李军2,李尚泽3,**()
1. 西藏大学医学院 拉萨 850000
2. 武汉爱博泰克生物科技有限公司 武汉 430072
3. 武汉大学中南医院　武汉　430071
Human CT55 Protein Prokaryotic Expression and Its Production of Monoclonal Antibody
Jian-wei REN1,Jun LI2,Shang-ze LI3,**()
1. Tibet University Medical College, Lasha 85000, China
2. ABclonal, Wuhan 430072, China
3. Zhongnan Hospital of Wuhan University, Wuhan　430071, China
 全文: PDF(1141 KB)   HTML
摘要：

为制备Cancer testis 55(CT55)单克隆抗体,需构建带有人源CT55片段的原核表达质粒,把该质粒转化Rosetta感受态进行原核表达并得到目的蛋白,蛋白质被纯化后免疫6周雌性BALB/c小鼠。按传统的单克隆抗体的制备方法,取小鼠脾细胞与骨髓瘤细胞(sp2/0)进行融合,经ELISA方法筛选及两次连续亚克隆,共获得多株能稳定分泌抗 CT55蛋白单克隆抗体的杂交瘤细胞,如3D8B7B12、4C8E1C9、3D8C10G9等。ELISA及Western blot(WB)分析结果表明,筛选的细胞株均能产生单克隆抗体,且该抗体均分别能与原核表达及真核表达的CT55蛋白发生特异性结合。单克隆抗体可用于免疫荧光试验,且与P53发生互作的荧光主要位于细胞核边缘。结果表明,成功制备了针对人源CT55 蛋白的单克隆抗体。CT55蛋白单克隆抗体的制备为今后肝癌、胃癌、结肠癌等癌症的快速的病原学诊断以及 CT55蛋白的结构和功能研究奠定了物质基础。
关键词： CT55;  原核表达;  单克隆抗体    
Abstract:

In order to prepare the monoclonal antibody of Cancer testis 55 (CT55), the prokaryotic expression plasmid with human CT55 fragment was constructed first, and the plasmid was expressed in the Rosetta competent cell. The target protein was purified and immunized for 6 weeks young female BALB/c mice. According to the preparation method of the conventional monoclonal antibody, the spleen and myeloma cells (sp2/0) of mice was prepared to fuse together. The hybridoma cell lines were obtained by indirect ELISA method and 2 successive subclones, such as 3D8B7B12, 4C8E1C9 and 3D8C10G9. The results of ELISA and Western blot analysis showed that the screened cell lines could produce monoclonal antibodies, and the antibody could specifically recognize the prokaryotic expression and eukaryotic expression of CT55 protein. Specific antibodies can be used in immunofluorescence test, and fluorescence is mainly located at the edge of nucleus. The results showed that monoclonal antibodies against human CT55 protein were successfully prepared. The preparation of CT55 protein monoclonal antibody has laid the material foundation for the rapid etiological diagnosis of cancer of liver cancer, gastric cancer and colon cancer, and the structure and function of CT55 protein.
Key words: CT55    Prokaryotic expression    Monoclonal antibody
收稿日期: 2018-06-23 出版日期: 2018-12-06
ZTFLH:  Q816  
基金资助: * 武汉大学青年教师资助项目(2042018kf0076)
通讯作者: 李尚泽     E-mail: 342883635@qq.com
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引用本文:

任建委,李军,李尚泽. 人源CT55蛋白原核表达及单克隆抗体的制备 *[J]. 中国生物工程杂志, 2018, 38(11): 1-8.

Jian-wei REN,Jun LI,Shang-ze LI. Human CT55 Protein Prokaryotic Expression and Its Production of Monoclonal Antibody. China Biotechnology, 2018, 38(11): 1-8.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20181101    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I11/1

图1  CT55基因克隆全过程
图2  CT55-H蛋白的诱导条件摸索和蛋白质纯化
图3  免疫小鼠血清效价测定
图4  细胞融合
1号 1 2 3 4 5 6 7 8 9 10 11 12
A 1.446 1.493 1.482 1.678 1.555 1.626 1.543 1.428 1.415 0.668 1.421 1.192 450
B 1.732 1.266 1.516 1.689 1.454 1.39 1.315 1.521 1.374 1.103 1.391 1.358 450
C 1.724 1.517 1.893 1.785 1.954 1.641 1.593 1.832 1.867 1.46 1.614 1.476 450
D 1.713 1.446 1.796 1.818 1.633 1.566 1.569 1.595 1.615 1.53 1.442 1.467 450
E 1.498 1.705 1.451 1.802 1.658 1.484 1.423 1.463 1.415 1.292 1.503 1.267 450
F 1.879 1.515 1.429 1.636 1.541 1.716 1.498 1.455 1.43 1.607 1.555 1.378 450
G 1.589 1.604 1.412 1.545 1.707 1.572 1.397 1.521 1.368 1.311 1.191 0.059 450
H 0.851 1.04 1.093 0.925 1.189 0.963 0.602 0.841 0.886 0.738 0.862 0.599 450
2号 1 2 3 4 5 6 7 8 9 10 11 12
A 1.427 1.335 1.329 1.303 1.193 1.199 1.292 1.301 1.176 1.243 1.277 1.348 450
B 1.471 1.485 1.287 1.595 1.39 1.374 1.402 1.318 1.399 1.267 0.834 1.041 450
C 1.668 1.396 1.428 1.416 1.734 1.446 1.443 1.423 1.431 1.502 1.36 1.289 450
D 1.397 1.463 1.411 1.723 1.463 1.382 1.442 1.339 1.251 1.8 1.262 1.046 450
E 1.551 1.386 1.436 1.502 1.424 1.65 1.433 1.523 1.408 1.571 1.121 0.858 450
F 1.607 1.635 1.619 1.491 1.593 1.523 1.438 1.843 1.39 1.394 1.236 1.531 450
G 1.45 1.549 1.587 1.465 1.476 1.616 1.436 1.631 1.653 1.374 1.249 1.331 450
H 1.575 1.778 1.524 1.571 1.517 1.618 1.315 1.621 1.644 1.56 1.392 1.533 450
3号 1 2 3 4 5 6 7 8 9 10 11 12
A 1.068 1.639 1.503 1.411 1.477 1.295 1.391 1.512 1.541 1.167 1.314 0.771 450
B 1.044 1.277 1.249 1.274 1.26 1.332 1.319 1.248 1.398 1.343 1.849 1.362 450
C 1.392 1.507 1.444 1.486 1.583 1.456 1.482 1.578 1.291 0.579 1.377 1.43 450
D 1.372 1.473 1.306 1.506 1.912 1.517 1.513 1.987 1.763 1.599 2.026 1.568 450
E 1.188 1.12 1.364 1.523 1.428 1.691 1.443 1.786 1.464 1.574 1.611 1.351 450
F 1.296 1.342 1.249 1.345 1.543 1.013 1.164 1.55 1.206 0.907 0.895 1.15 450
G 1.422 1.364 1.365 1.403 1.646 1.233 1.387 1.532 1.439 1.168 1.447 1.297 450
H 1.371 1.378 1.267 1.328 1.493 1.253 1.252 1.306 1.422 1.249 1.33 1.35 450
4号 1 2 3 4 5 6 7 8 9 10 11 12
A 1.282 1.336 1.337 1.503 1.538 1.512 1.565 1.681 1.057 1.381 1.475 1.209 450
B 1.308 1.893 1.544 1.606 1.695 1.709 1.483 1.833 1.693 1.52 1.846 1.528 450
C 1.187 1.831 1.781 1.789 1.868 1.659 1.673 1.687 1.879 1.739 2.09 1.575 450
D 1.444 1.426 1.585 1.409 1.79 1.606 1.423 1.497 0.352 0.056 0.109 0.118 450
E 1.297 1.692 1.55 1.767 1.629 1.634 1.724 1.845 1.731 1.504 1.869 1.684 450
F 1.388 1.495 1.442 1.475 1.742 1.979 1.604 1.699 1.506 1.48 1.632 1.613 450
G 1.367 1.623 1.485 1.63 1.8 1.704 1.706 2.269 1.959 1.681 1.873 1.592 450
H 1.379 1.48 1.503 1.533 2.014 1.59 1.558 1.897 1.643 1.617 1.751 1.44 450
5号 1 2 3 4 5 6 7 8 9 10 11 12
A 1.296 1.304 1.27 1.212 1.389 1.221 1.428 1.267 1.267 1.11 1.339 0.97 450
B 1.408 1.516 1.226 1.339 1.251 1.27 1.295 1.237 1.184 1.125 1.017 0.837 450
C 1.566 1.324 1.412 1.332 1.366 1.307 1.365 1.435 1.261 0.789 1.037 0.819 450
D 1.325 1.379 1.267 1.287 1.293 1.127 1.425 1.397 1.138 0.611 1.038 0.533 450
E 1.463 1.251 1.183 1.291 1.229 1.25 1.627 1.351 1.181 1.228 1.083 1.5 450
F 1.44 1.236 1.206 1.26 1.506 1.164 1.141 1.164 1.131 0.938 1.07 0.982 450
G 1.446 1.411 1.194 1.476 1.341 1.213 1.277 1.201 1.508 1.076 1.577 0.956 450
H 1.128 1.555 1.335 1.388 1.338 1.241 1.293 1.189 1.353 1.165 1.158 0.059 450
表1  采用ELISA方法筛选阳性克隆细胞株
图5  第二次亚克隆Western blot检测结果
图6  排除其他蛋白质及标签的影响
图7  CT55蛋白与P53蛋白的荧光共定位
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