《中国畜牧兽医》 ›› 2019, Vol. 46 ›› Issue (8): 2438-2445.doi: 10.16431/j.cnki.1671-7236.2019.08.030

• 预防兽医 • 上一篇    下一篇

鼠源CD40L的原核表达及其对河豚毒素半抗原免疫增强效果分析

潘悦, 曹旗, 任洪林, 胡盼, 李岩松, 周玉, 柳增善, 卢士英   

  1. 吉林大学人兽共患病研究所, 人兽共患病教育部重点实验室, 长春 130062
  • 收稿日期:2019-01-24 出版日期:2019-08-20 发布日期:2019-08-17
  • 通讯作者: 卢士英 E-mail:lushiying1129@163.com
  • 作者简介:潘悦(1992-),女,山东日照人,硕士,研究方向:动物性食品安全,E-mail:1312261957@qq.com
  • 基金资助:

    吉林大学省校共建项目(SXGJSF2017-6)

Prokaryotic Expression of Murine CD40L and Its Immunopotentiating Effect on Tetrodotoxin Hapten

PAN Yue, CAO Qi, REN Honglin, HU Pan, LI Yansong, ZHOU Yu, LIU Zengshan, LU Shiying   

  1. Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun 130062, China
  • Received:2019-01-24 Online:2019-08-20 Published:2019-08-17

摘要:

试验旨在通过大肠杆菌表达系统表达鼠源重组CD40L蛋白,探讨其对河豚毒素(tetrodotoxin,TTX)人工完全抗原在免疫BALB/c小鼠过程中的免疫增强作用。用Trizol试剂提取BALB/c小鼠脾脏总RNA并反转录成cDNA,根据CD40L CDS区设计引物,PCR扩增目的基因,构建pGEX4T-1重组载体,进行原核表达,并纯化重组CD40L蛋白;根据曼尼希反应原理,用甲醛法制备TTX免疫原TTX-BSA和检测原TTX-OVA;以人工重组蛋白CD40L佐剂组为试验组,弗氏佐剂组作为对照组,免疫BALB/c小鼠,用ELISA方法结合SPSS 19.0软件分析各组免疫效果,探讨重组CD40L蛋白在TTX-BSA免疫过程中对免疫效果的影响。结果显示,本试验成功扩增了783 bp的CD40L目的基因,原核表达并纯化了融合GST标签的55 ku鼠源CD40L重组蛋白;与人工制备的TTX-BSA协同免疫小鼠试验结果显示,在免疫初期与弗氏佐剂相比,CD40L具有极显著的免疫增强效果(P<0.01)。综上所述,重组蛋白CD40L与TTX-BSA完全抗原协同免疫小鼠,在免疫初期CD40L具有增强机体对半抗原的应答强度的作用,为进一步开发适于小分子半抗原抗体高效制备的免疫增强佐剂奠定基础。

关键词: CD40L; 佐剂; 河豚毒素; 免疫效果

Abstract:

The aim of this study was to express recombinant protein CD40L from mice by E.coli expression system, and investigate its immunopotentiating effect on tetrodotoxin (TTX) artificial complete antigen in BALB/c mice.Total RNA from spleen of BALB/c mice was extracted by Trizol reagent and retranscribed into cDNA.Primers were designed according to CD40L CDS region, and the target gene was amplified by PCR.The recombinant vector pGEX4T-1 was constructed for prokaryotic expression, and the recombinant CD40L protein was purified.TTX-BSA and TTX-OVA were prepared by formaldehyde method according to Mannich reaction principle.The adjuvant of artificial recombinant protein CD40L was used as the experimental group and the adjuvant of Freund's was used as the control group.BALB/c mice were immunized.The immune effects of each group was analyzed using ELISA method combined with SPSS 19.0 software to explore the effect of recombinant CD40L protein on the immune effect during TTX-BSA immunization.This experiment successfully amplified 783 bp CD40L gene, prokaryotic expression and purification of GST-tagged 55 ku mice CD40L recombinant protein.Compared with TTX-BSA synergistic immunization in mice, CD40L had extremely significant immune enhancement effect at the initial stage of immunization (P<0.01) than Freund's adjuvant.In conclusion, the recombinant protein CD40L and TTX-BSA complete antigen synergistically immunized mice could enhance the body's response to hapten at the initial stage of immunization, which laid a foundation for further development of immunopotentiation adjuvants suitable for the efficient preparation of small hapten antibodies.

Key words: CD40L; adjuvant; tetraodotoxin; immune effect

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