Anti-hK7 Polyclonal Antibody Generation And Preliminary Investigation Of HK7 Expression In Prostate Carcinoma And BPH Tissues

Background: Kallikreins are a subgroup of serine proteases with diverse physiological functions. Many Kallikrein genes are differentially expressed in various malignancies, especially in hormone-dependent malignancies. Prostate specific antigen (PSA; encoded by the KLK3 gene) is the best tumor marker for prostate cancer. KLK7 is a newly discovered human Kallikrein gene which shares a high degree of homology and is located adjacent to KLK3 genes on chromosome 19q13.3-4. In our previous experiments, KLK7 expression in co-cultured epithelial cells with fibroblast cells showed higher than that in non-cocultured epithelial cells. For further study on expression and function of hK7, it becomes the initial mission to generate antibody against hK7.Objective: To construct the recombinant vector pET-21a-KLK7, induce expression of the recombinant protein in E.coli BL21 (DE3), purify and identify fusion protein, and obtain rabbit anti-hK7 polycolonal antibody. Then to investigate the expression of hK7 in different prostate tissues (normal prostate, BPH and prostate carcinoma) to preliminarily research whether hK7 is differentially expressed and its significance.Methods:1. First, we amplified KLK7 gene by PCR. The amplifying segments and the pET-21a vector were both double digested by restriction enzyme Ndeâ… and Xhoâ… , and then they were linked by T4 DNA ligase.2. The pET-21a-KLK7 recombinant plasmid was amplified by transformation into competent E.coli BL21 (DE3), and colony PCR was used to screen and identify positive clone. Then we extracted plasmid from positive clone, and the recombinant plasmid was detected by sequencing.