التفاصيل البيبلوغرافية
| العنوان: |
布鲁氏菌分泌蛋白 BspA 和 BspB 真核表达载体的构建及在胚胎滋养层细胞中的表达. (Chinese) |
| العنوان البديل: |
Construction of Eukaryotic Expression Vectors of Brucella Secretory Proteins BspA and BspB and Expression in Embryonic Trophoblast Cells. (English) |
| المؤلفون: |
王书利, 魏淑娟, 李梦含, 郑 好, 司丽芳, 李志强 |
| المصدر: |
Journal of Henan Agricultural Sciences; 2022, Vol. 51 Issue 8, p143-149, 7p |
| مصطلحات موضوعية: |
LACTATE dehydrogenase, GENETIC vectors, PROTEIN expression, SEQUENCE analysis, WESTERN immunoblotting |
| الملخص (بالإنجليزية): |
To study the role of Brucella secretory proteins BspA and BspB, two pairs of primers were designed according to BspA and BspB gene sequences of B. abortus S2308 from GenBank, and S2308 genome was used as template to amplify BspA and BspB genes by PCR. The fragments were cloned into pEGFP‑N1 vector to obtain eukaryotic vectors pEGFP‑BspA and pEGFP‑BspB. After identification by enzyme digestion and sequencing analysis, LipofectamineTM 2000 was used to transfect the eukaryotic vectors into embryonic trophoblast cells（HPT‑8). BspA and BspB protein expressions were detected by Western blot（WB). The secretion level of cytokines was detected by ELISA kit, and the level of cytotoxicity was detected by lactate dehydrogenase（LDH）cytotoxicity detection kit. The results showed that the full length of BspA gene was 576 bp and BspB gene was 564 bp. Eukaryotic vectors were verified by restriction enzyme EcoR Ⅰ and Xba Ⅰ digestion, and sequencing analysis showed that the homology was 100% between the sequencing information and sequence published by GenBank. WB detection results showed that the transfection groups of pEGFP‑BspA and pEGFP‑BspB dispalyed bands at 70 ku and 68 ku respectively, which was completely consistent with the expected results, indicating that BspA and BspB proteins could be expressed in HPT‑8 cells. Cytokines test results showed that BspA and BspB proteins could induce HPT‑8 cells to secrete IFN‑γ, IL‑2, IL‑4 and IL‑5. BspA and BspB proteins were non‑toxic to HPT‑8 cells. In summary, pEGFP‑BspA and pEGFP‑BspB eukaryotic expression vectors were successfully constructed, and BspA and BspB proteins could be expressed in HPT‑8 cells and induce cytokines secretion. [ABSTRACT FROM AUTHOR] |
| Abstract (Chinese): |
为研究布鲁氏菌分泌蛋白 BspA和 BspB的作用，以布鲁氏菌 S2308基因组为模板，根据 GenBank 登录的BspA和BspB基因序列设计引物，利用PCR扩增BspA和BspB基因片段，并将其克隆至pEGFP-N1 载体，获得真核表达载体 pEGFP-BspA和 pEGFP-BspB。真核表达载体经酶切和测序分析鉴定后，利用 LipofectamineTM 2000脂质体转染至胚胎滋养层细胞（HPT-8)，采用 Western blot（WB）检测 BspA 和 BspB 蛋白的表达，应用ELISA试剂盒检测细胞因子的分泌水平，利用乳酸脱氢酶（LDH）细胞毒性检测试剂盒 检测细胞毒性水平。结果显示，BspA基因大小为576 bp，BspB基因大小为564 bp，真核表达质粒经限制 性内切酶EcoR Ⅰ和Xba Ⅰ酶切验证正确，酶切产物经测序分析，显示与GenBank公布序列的同源性为 100%；WB检测结果显示，pEGFP-BspA和pEGFP-BspB转染组分别在70 ku和68 ku处出现条带，与预期 结果相符，表明 BspA 和 BspB 蛋白能在 HPT-8细胞中表达；细胞因子检测结果显示，BspA 和 BspB 蛋白 可诱导HPT-8细胞分泌IFN-γ、IL-2、IL-4和IL-5；细胞毒性检测结果显示，BspA和BspB蛋白对细胞无 毒性。综上，成功构建了真核表达载体pEGFP-BspA和pEGFP-BspB，证实其能在HPT-8细胞中表达，并 能诱导细胞因子分泌。 [ABSTRACT FROM AUTHOR] |
|
Copyright of Journal of Henan Agricultural Sciences is the property of Editorial Board of Journal of Henan Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) |
| قاعدة البيانات: |
Complementary Index |
