Chen Yuanyuan, Yang Wei, Xu Chuang
College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing High-tech Industrial Development Zone, Daqing 163319, PR China.
J Vet Res. 2017 Dec 6;61(2):203-209. doi: 10.1515/jvetres-2017-0026. eCollection 2017 Jun.
Vascular cell adhesion molecule 1 (VCAM-1) is a member of Ig superfamily. The aim of this study was to prepare highly specific polyclonal antibodies against bovine VCAM-1 and to evaluate the expression of VCAM-1 in the mammary lymph nodes of cows with subclinical mastitis.
The VCAM-1 gene was cloned from bovine Peyer's patches and inserted into the pGEX-4T-1 and pET-28a vectors. The recombinant plasmids pGEX-4T-1/VCAM-1 and pET-28a/VCAM-1 were transferred into BL21 and the recombinant strains were induced by isopropyl-D-thiogalactoside to produce fusion proteins tagged with polyhistidine (His) and glutathione S-transferase (GST), respectively. The expressed fusion proteins His-VCAM-1 and GST-VCAM-1 were identified by SDS-PAGE and Western blot. His-VCAM-1 protein was used as an antigen to immunise Wistar rats and polyclonal antibody serum against VCAM-1 was obtained.
The serum titre tested by indirect ELISA was 128,000 using GST-VCAM-1 as the well coating antigen. Western blots indicated that the antibody recognised recombinant VCAM-1 protein as well as endogenous VCAM-1. In addition, using qPCR and Western blot, VCAM-1 mRNA and protein expression levels were measured in dairy cows with subclinical mastitis. It was demonstrated that VCAM-1 levels in the mammary lymph nodes of the cows were significantly higher than those from healthy controls (P < 0.05).
These results are to our knowledge the first report that VCAM-1 expression in the mammary lymph nodes is elevated in dairy cows with subclinical mastitis.
血管细胞黏附分子1（VCAM-1）是免疫球蛋白超家族的成员。本研究旨在制备针对牛VCAM-1的高特异性多克隆抗体，并评估VCAM-1在亚临床型乳腺炎奶牛乳腺淋巴结中的表达。
从牛派伊尔氏淋巴集结中克隆VCAM-1基因，并将其插入pGEX-4T-1和pET-28a载体。将重组质粒pGEX-4T-1/VCAM-1和pET-28a/VCAM-1转入BL21，用异丙基-β-D-硫代半乳糖苷诱导重组菌株分别产生带有多组氨酸（His）和谷胱甘肽S-转移酶（GST）标签的融合蛋白。通过SDS-PAGE和蛋白质免疫印迹法鉴定表达的融合蛋白His-VCAM-1和GST-VCAM-1。用His-VCAM-1蛋白作为抗原免疫Wistar大鼠，获得抗VCAM-1的多克隆抗体血清。
以GST-VCAM-1作为包被抗原，通过间接ELISA检测血清效价为128,000。蛋白质免疫印迹表明，该抗体可识别重组VCAM-1蛋白以及内源性VCAM-1。此外，采用实时荧光定量PCR和蛋白质免疫印迹法，检测了亚临床型乳腺炎奶牛中VCAM-1的mRNA和蛋白表达水平。结果表明，奶牛乳腺淋巴结中VCAM-1水平显著高于健康对照（P < 0.05）。
据我们所知，这些结果首次报道了亚临床型乳腺炎奶牛乳腺淋巴结中VCAM-1表达升高。
