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Preparation of Anti-he4 Monoclonal Antibodies for Detection of Ovarian Cancer

Author: ZuoHaoFei
Tutor: SongHaiFeng;FanLiBin
School: Anhui Medical University,
Course: Cell Biology
Keywords: Ovarian cancer HE4 In vivo electroporation DNA immunization Hybridoma Monoclonal antibody
CLC: R737.31
Type: Master's thesis
Year: 2010
Downloads: 146
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Abstract


Ovarian cancer is a serious threat to the majority of women disease that, due to the incidence of part of occult, early symptoms, ovarian cancer mortality rate ranks first in gynecological malignancies, 70% to 80% of patients with ovarian cancer found late, the five-year survival rate was 20% of the patients with early 5-year survival of up to 70% to 90%, so early ovarian cancer diagnosis has important significance to improve the cure rate, which is currently gynecologic oncology research focuses on the hot. Simple and non-invasive detection of tumor markers in cancer screening, diagnosis and guide treatment, prognosis evaluation has a very broad application prospects. At present, only the cancer antigen 125 (cancer antigen 125, CA125) detection is widely used in the clinical diagnosis of ovarian cancer, a large number of studies have confirmed that CA125 sensitivity and specificity is not high, the low rate of early diagnosis of ovarian cancer. Human epididymis protein 4 (human epididymis protein 4, HE4) is a new ovarian cancer tumor markers, was originally found in the epididymal epithelium, protease inhibitors, but the specific function is unknown. HE4 in ovarian cancer with high expression or low expression in normal tissues, its sensitivity in the diagnosis of ovarian cancer was significantly higher than CA125, and can better distinguish between benign and malignant tumors, a large number of studies have confirmed that HE4 is the detection of early ovarian one of the best markers of cancer. Thus prepared for improving the survival rate of patients with ovarian cancer, ovarian cancer early diagnostic reagents for HE4 significance. The traditional the antibody preparation method usually purified protein to immunize animals, but the separation and purification of proteins time-consuming and difficult to get a good product, and sometimes even with the target protein, low immunogenicity (such as some prokaryotic expression protein), it is difficult to obtain the desired immune effect. Electroporation auxiliary DNA immunization can overcome these shortcomings. The instantaneous electric cell membrane can produce recoverable microporous exogenous gene into the target tissue or organ in the electric field force through the porous, and direct expression of the target antigen in the tissue or organ, trigger an immune response. DNA after cellular uptake, Gene half-life extension, sustainable expression of the target protein, enhanced lymphocyte memory persistence; addition, electroporation cause local tissue inflammatory response, so that a large number of inflammatory cell infiltration, and to improve the antigen presenting efficiency, therefore, the electric perforation secondary DNA immunization can obtain high titers of antibodies. In this study, using RT-PCR the HE4 gene coding sequence obtained from the tissue of patients with ovarian cancer, HE4 gene was cloned into the TA cloning the pMD19T carrier will be identified by sequencing subcloned to pPICZαA to and pcDNA3.1 () expression vector, and by PCR, double enzyme digestion and sequencing of their identification. The constructed expression plasmid transfected by electroporation of Pichia pastoris GS115 screening positive expression strain on the selection medium to induce the expression of HE4 recombinant protein further by ELISA and Western-blot identification of recombinant protein expression of HE4 pPICZαA-HE4. By examining the type of the methanol concentration, the pH, the induction time, the expression levels of biomass and induction temperature HE4 optimization of HE4 induced expression conditions. Containing of HE4 recombinant protein of yeast culture supernatant using a nickel affinity chromatography purification. On the other hand, we vivo electroporation-assisted pPICZaA-HE4 and pcDNA3.1-HE4 were immunized BALB / c mice, cell fusion to prepare monoclonal antibodies by B lymphocyte hybridoma technique, after repeated cloning culture, HE4 mAb specific secretion of mouse anti-human hybridoma cell lines screened. Western-blot analysis of Ig subtypes and mAb epitope analysis to identify the biological characteristics of mAb. The results showed that the success obtained from ovarian cancer HE4 gene and constructed two eukaryotic expression vector pcDNA3.1-HE4 and pPICZαA-HE4; build Pichia yeast secretory expression of HE4 protein, recombinant protein. glycosylation and to have a certain spatial conformation; the best of HE4 recombinant protein inducing conditions: pH4.0, 2% methanol, 2 times the amount of bacteria, the temperature 26 ° C, induced by 12h; from 1L culture supernatant, may be approximately 10 mg of the HE4 recombinant protein; purified monoclonal antibody preparation, the two sustained, stable hybridoma cell lines secreting anti HE4 mAb, named 1-7-C-12-C, two single anti-culture supernatant antibody titers of 1:160 and 1:320, respectively, ELISA and Western-blot results showed: two mAbs obtained in this study can specifically identify the HE4 protein of the native conformation, the HE4 protein the same epitope two mAb Ig subclass are IgM, light chains are lambda chain. These results indicate that HE4 gene was successfully cloned into two eukaryotic expression vector pcDNA3.1-HE4 and pPICZαA-HE4; successfully constructed expression of the the HE4 protein of yeast engineered bacteria, the spatial conformation of HE4 recombinant proteins, and 2 secreting anti HE4 mAb hybridoma cell lines, laid the foundation for ovarian cancer early diagnosis and HE4 protein functional studies.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Female genital tumors > Ovarian tumors
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