Kaur Navneet, Sunil Kumar B V, Mahajan Kanika, Singh Satparkash
a School of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University , Ludhiana , India.
J Immunoassay Immunochem. 2016;37(5):515-26. doi: 10.1080/15321819.2016.1162800.
All four members of the tissue inhibitors of metalloproteinase (TIMP) family have been reported to be over-expressed in breast cancer cells in vitro. Dysregulation of TIMP-4 expression predicts poor prognosis in cancers. The present study evaluated the association of the expression levels of TIMP-4 with mammary tumor development in dogs, measured by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mammary tissue samples were collected from healthy canine mammary gland and from tumor subjects. TIMP-4 expression was found to be upregulated (5.856 times) in complex canine mammary carcinomas. Also, TIMP-4 mature peptide was expressed heterologously in E. coli. The recombinant protein was purified by Ni- NTA affinity chromatography and further confirmed by western blotting. The rTIMP-4 was found to be functionally active and could inhibit matrix metalloproteinase 11(MMP-11) activity. Immunization of mice with rTIMP-4 resulted in increased antigen specific serum antibody titer, and this serum could be suitably used to detect and quantify the protein in sera of dogs with mammary tumors. TIMP-4 could act as a marker of canine mammary tumors. To the best of our knowledge, this is the first report of heterologous expression of TIMP-4 from complex canine mammary carcinomas.
据报道，金属蛋白酶组织抑制剂（TIMP）家族的所有四个成员在体外乳腺癌细胞中均有过表达。TIMP-4表达失调预示着癌症预后不良。本研究通过实时定量逆转录聚合酶链反应（qRT-PCR）评估了TIMP-4表达水平与犬乳腺肿瘤发生之间的关联。从健康犬乳腺组织和肿瘤组织中采集乳腺组织样本。发现TIMP-4在复杂型犬乳腺癌中表达上调（5.856倍）。此外，TIMP-4成熟肽在大肠杆菌中进行了异源表达。重组蛋白通过镍-氮川三乙酸亲和层析法纯化，并通过蛋白质印迹进一步确认。发现重组TIMP-4（rTIMP-4）具有功能活性，能够抑制基质金属蛋白酶11（MMP-11）的活性。用rTIMP-4免疫小鼠导致抗原特异性血清抗体滴度升高，并且该血清可适用于检测和定量患有乳腺肿瘤犬的血清中的蛋白质。TIMP-4可作为犬乳腺肿瘤的标志物。据我们所知，这是关于复杂型犬乳腺癌中TIMP-4异源表达的首次报道。
