| Breast cancer is the most common malignancy which seriously threats to the health of women. In recent years, the incidence of breast cancer in Chinese women is rising, especially in economically developed areas [1]. Now the etiopathogenisis of breast cancer is not very clear. Mammary gland is the organ responsing for major hormones. Endocrine dyscrasia may play an important role in breast genesis of breast cancer [2]. With the research of function of hormone and its receptor in breast tissue, and the development of molecular mechanism in breast cancer genesis and progression, prolactin (PRL) and prolactin receptor (PRLR) is reemphasized by many laboratories. PRL could influence mammary cell from cell division, cell form and excretion. Recent studies show that PRL and PRLR are at a high level in neoplastic tissues and breast cancer cell. PRL shows some characters of cytokines through its receptor. PRLR could activate the downstream JAK and STAT signaling pathways, and induce the genesis of breast cancer [3]. Therefore, inhibition or blocking of PRLR expression may be a new method for breast cancer biotherapy.Objective: Blood was collected from breast cancer patients to structure large phage-display Fab library originated from breast cancer patients. This phage-display library was panned by hPRLR ECD to obtain the corresponding humanized Fab antibody . Methods: (1) Blood was collected from 24 breast cancer patients left over from clinical diagnostic specimens. After separating lymphocytes from the blood, RNA was extracted. The variable region gene (VH, VLκand VLλ) were amplified from the cDNA by RT-PCR. After amplifying the constant region gene, Fab gene was amplified by overlap PCR. The Fab gene was plug into the phage vector pComb3XSS. The vector was electro-transformed into E.coli XL1-Blue. After phage infection, we harvested the natural human Fab phage antibody library originated from breast cancer patients. And then, test the size of the library.(2)The hPRLR ECD was used as the antigen to pan the library. After several rounds of panning, we choose 50 clones form the last round output. Test the affinity of the clone to the antigen by ELISA. Distill the vector of the positive recombinant phages and transform into E.coli TOP10. The soluble Fab antibodies were produce after inducement. And then, we did Western-blot and Fab-ELISA to evaluate the clone initiatively .Results: (1) The Fab antibody fragment with good diversity was amplified by overlap PCR. The Fab gene was plug into the phage vector pComb3XSS. After 100 times electro-transformation and phage infection, we constructed a human naive Fab library originated from breast cancer with the diversity of 1.0×109.(2) Panned the library by soluble hPRLR ECD. The sixth eluting we get the phage antibody have 2.4×102 than the first eluting. The phage anti-hPRLR antibodies(X8,X10,X19,X30) of were identified by ELISA. X8,X10,X19 was analyzed to be the same clone by sequencing (hereinafter collectively referred to as X19) . The vector of X19 was transformed into E.coli. TOP10. The soluble Fab protein was produce after inducement. Two protein bands were found at about 29kD and 25kD by SDS-PAGE and Western-blot. It also has a positive reaction in Fab-ELISA test.Conclusion: (1) The Fab antibody fragment was amplify with good diversity. A human Fab large phage-display library originated from breast cancer patients was successfully constructed, which have a diversity of 1.0×109. This library may have superiority in panning the antibody that has more to do with breast cancer. (2)After six times'panning, one antibody was found, which is called X19. And it may useful to study on the PRLR antagonist. |
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