Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Dec 6;61(2):203-209.
doi: 10.1515/jvetres-2017-0026. eCollection 2017 Jun.

Expression of Vascular Cell Adhesion Molecule 1 (VCAM-1) in the Mammary Lymph Nodes of Cows with Subclinical Mastitis

Yuanyuan Chen  1 , Wei Yang  1 , Chuang Xu  1
Affiliations

Expression of Vascular Cell Adhesion Molecule 1 (VCAM-1) in the Mammary Lymph Nodes of Cows with Subclinical Mastitis

Yuanyuan Chen et al. J Vet Res. .

Abstract

Introduction: 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.

Material and methods: 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 Escherichia coli 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.

Results: 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).

Conclusion: 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.

Keywords: VCAM-1; dairy cows; mammary lymph nodes; polyclonal antibody; subclinical mastitis.

PubMed Disclaimer

Conflict of interest statement

Conflict of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article.

Figures

Fig. 1
Fig. 1
The PCR product of VCAM-1 gene. M – DL2000 Marker; 1 – M gene
Fig. 2
Fig. 2
Enzyme digestion profile of pGEX-4T-1-VCAM-1. M – DL2000 Marker; 1, 2 – Identification of pGEX-4T-1-VCAM-1 with Eco RI and XhoI digestion
Fig. 3
Fig. 3
Enzyme digestion profile of pET-28a-VCAM-1. M – DL2000 Marker; 1, 2 – Identification of pET-28a-VCAM-1 with with Eco RI and XhoI digestion
Fig. 4
Fig. 4
Expression of GST-VCAM-1 analysed by 12% SDS–PAGE. Lane1 – whole lysate of cells harbouring pGEX-4T-1-VCAM-1 without IPTG induction; Lanes 2 and 3 – whole lysate of cells harbouring pGEX-4T-1-VCAM-1 after IPTG induction
Fig. 5
Fig. 5
Expression of His-VCAM-1 analysed by 12% SDS–PAGE. Lane 1 – whole lysate of cells harbouring pET-28a-VCAM-1 without IPTG induction; Lane 2 – whole lysate of cells harbouring pET-28a-VCAM-1 after IPTG induction
Fig. 6
Fig. 6
Western blot analysis of recombinant VCAM-1-GST protein. M – prestained protein marker; 2 – anti-GST antibody as the first antibody
Fig. 7
Fig. 7
Western blot analysis of recombinant VCAM-1-His protein. M – prestained protein marker; 2 – anti-His antibody as the first antibody
Fig. 8
Fig. 8
Western blot analysis of VCAM-1-His Polyclonal antibody to identify recombinant VCAM-1-GST protein. M – prestained protein marker; 1 – GST; 2 – VCAM-1-GST
Fig. 9
Fig. 9
Detection of VCAM-1 gene transcription differences in lymphoid tissues from subclinical mastitis and healthy dairy cows by real time-PCR
Fig. 10
Fig. 10
Detection of VCAM-1/ MAdCAM-1 gene expression differences in lymphoid tissues from subclinical mastitis and healthy cows by Western blot
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Ahrens R., Waddell A., Seidu L., Blanchard C., Carey R., Forbes E., Lampinen M., Wilson T., Cohen E., Stringer K., Ballard E., Munitz A., Xu H., Lee N., Lee J.J., Rothenberg M.E., Denson L., Hogan S.P.. Intestinal macrophage/epithelial cell-derived CCL11/Eotaxin-1 mediates eosinophil recruitment and function in pediatric ulcerative colitis. J Immunol. 2008;10:7390–7399. - PMC - PubMed
    1. Bai R., Bai H., Kuse M., Ideta A., Aoyagi Y., Fujiwara H., Okuda K., Imakawa K., Sakurai T.. Involvement of VCAM1 in the bovine conceptus adhesion to the uterine endometrium. Reproduction. 2014;2:119–127. - PubMed
    1. Bar D., Tauer L.W., Bennett G., Gonzalez R.N., Hertl J.A., Schukken Y.H., Schulte H.F., Welcome F.L., Gröhn Y.T.. The cost of generic clinical mastitis in dairy cows as estimated by using dynamic programming. J Dairy Sci. 2008;6:2205–2214. - PubMed
    1. Carvalho A.T., Elia C.C., de Souza H.S., Elias P.R., Pontes E.L., Lukashok H.P., de Freitas F.C., Lapa e Silva J.R.. Immunohistochemical study of intestinal eosinophils in inflammatory bowel disease. J Clin Gastroenterol. 2003;2:120–125. - PubMed
    1. Clahsen T., Pabst O., Tenbrock K., Schippers A., Wagner N.. Localization of dendritic cells in the gut epithelium requires VCAM-1. Clin Immunol. 2014;1:74–84. - PubMed

LinkOut - more resources