Red Cloning And Antibody Preparation Prostate Cancer, The Biological Model Of The Establishment And Analysis

λ-phage Red recombinase mediates the high efficiency homologous recombination in vivo which is crucial in reforming transgenic animal vectors and bacterial hereditary properties, etc.In oder to study the interaction among the expression of exo,beta and gam, genes were introduced into prokaryotic expression vetor pEGKG and pET28a, then transformed into competent E.coli BL21(DE3) and DH5α, respectively. Induced by IPTG, corresponding His or GST tag fused soluble expression proteins were obtained in E.coli. Exo Proteins were initially purified with anion-exchange column, then his-tag fusion proteins were purified with chromatographic column filled with Ni-NTA and GST-fusion proteins with chromatographic column filled with glutathione Sepharose 4B amboceptor. Antigens for immuning animals were prepared by NC membrane combined techniques with these two-step purified proteins. Verified by ELISA and western blottings, we acquire polyclonal antibodies whose titers would attain as high as 1∶12800 and specifically combine Exo antigen.With this process, it's easier to gain highly purified antigens and the affinity of relevant polyclonal antibodies are strong. polyclonal antibodies preparation is easily operated compared to other methods. Prostate cancer(PCa)is one of the most common malinant tumor in male reproductive system. The incidence of prostate cancer occupies the first place of all malignant tumors in the United States, second only to lung cancer mortality. Since the concealment and relatively long course of Pca bring difficulties to clinical research, it's meaningful to develop a model to mimic the histopathology of human PCa development. Several transgenic mice are used abroad at present, while little relevant research reports in China.Sternberg et. discovered the Cre recombinase in P1 Phage which encoding a 38 kDa protein, belongs toλintegrase gene superfamily, a site-specific recombinase that separately mediate the accurate recombination between Loxp sites. Cre / LoxP can be effective in vivo gene deletion, insertion, translocation and inversion. In oder to facilitate the development of prostate tissue-specific conditional gene targeting mice models of prostate cancer, Cre / LoxP system is used.In this study, we used PSP94, a prostate tissue-specific promoter that promote the expression of Cre recombinase to create PSP94-Cre transgenic mice. By microinjection,a 4.9 kb DNA fragment of PSP94-Cre were inject into mice's fertilized eggs and five positives in 20 offspring were obtained by genotyping .Prostate cancer is androgen-associated.The biological effects of androgen is androgen receptor (androgen receptor, AR)-mediated. AR play as a role of facilitating or inhibiting the growth of prostate cancer through regulating the transcription of target genes.Many studies found that, AR may play dual roles in the development of prostate cancer, it promotes the growth of prostate cancer cells in one hand and promotes the apoptosis of prostate cancer cells in the other. The role of AR in prostate cancer endocrine therapy resistance have been research focus. Even if PC3 prostate cancer cells are androgen-dependent and metastasis, they don't express AR.Using the DNA transfection technique,the vectors pCMV-Tag 2B-AR which carry AR gene were transfected into PC3 and a cell line that stably expresses AR was established. Follow by the detection of PC3-AR proliferation, we tend to investigate the affection of AR in hormone-independent cell proliferation.Preliminary studies showed that PC-1 gene expression promote prostate cancer cells growth and it would interact with AR on the protein level. To study the affection of PC-1 on AR transcriptional activity, LNCaP/C4-2 and PC3 (AR) prostate cancer models were used. A number of different domains of deletion mutants of PC-1 were cotransfected with PSA-LUC and we found that the N terminal 46 amino residues of PC-1 could decrease the transcriptional activity of AR. What's more, R1881 induces the epression of PC-1 and colocalize with AR in nucleus.