Preliminary Study On Expression In Prokaryotic Cells, Preparation And Application Of Polyclonal Antibody To Lung Cancer Metastasis-Related Protein 1 (LCMR1)

Objective: Lung Cancer Metastasis-Related Protein 1 ( LCMR1) is a new protein cloned by differential display PCRPD-PCR) in our laboratory. To explore biological function of LCMR1, this research aims at acquiring purified fusion protein of LCMR1 from prokaryotic cells to prepare LCMR1 antibody, and then detecting LCMR1 expressions in normal and tumor tissues of human.Methods: (1) The inducing condition of the pGEX-5T/LCMR1 prokaryotic expression vector was optimized to acquire stable expression of GST-LCMR1 in E.coli cells. The technique of affinity chromatography was employed to purify the fusion protein, which was then identified by western blot and MALDI-TOF-MS;(2) The antibody of LCMR1 was prepared by immunizing the New Zealand rabbit with the purified fusion protein or with the synthesized peptide;(3) The expression of LCMR1 in cells and tissues of human was detected by using the technique of tissue microarray and immunohistochemistry.Results: (1) The critical parameters of LCMR1 prokaryotic expression in E.coli were established: the inducing temperature was 20 ℃, the inducing time 4 hours and the dosage of IPTG 0.6mmol/L. The GST-LCMR1 fusion protein was successfully purified by affinity chromatography using glutathione-agarose resin, with the purity of aimed protein above 85%;(2) The LCMR1 antibody was successfully prepared by immunizing the rabbit with the purified fusion protein and with the synthesized peptide, and the antibody was of good quality with good titer and specificity;(3) The expression of LCMR1 was low in thyroid gland ,striated muscle and ovarian cancer, while high in the cell line of 95D and 95C, lung, stomach, liver, heart, colon, esophagus and prostate and their respective tumors. It was also found that LCMR1 was mainly located in thecellular cytoplasm of the cell lines of 95Dand 95C as well as the tissue of muscle and heart, but was mainly found in the cellular nucleus of other tissues.Conclusions: (1) The stable expression of GST-LCMRl was obtained by using E.coli system and a purification line of high efficiency was set up;(2) The LCMRl antibody was successfully acquired by employing two different methods;(3) The expression of LCMRl in human normal and tumor tissues was explored at the protein level, the result of which showed that the expression of LCMRl was high in many normal and tumor tissues of human. This demonstrated that LCMRl could be an important protein that is playing a vital role in the basic functioning of human life.