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The Expression of the Liver Specific F Protein and the Preparation of Monoclonal Antibody

Author: SongZuoLi
Tutor: LiuShuYe
School: Tianjin Medical University
Course: Clinical Laboratory Science
Keywords: F protein Liver Antibody Monoclonal Immunity
CLC: R392
Type: Master's thesis
Year: 2008
Downloads: 57
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Abstract

Liver disease, serious impact on human health, and have a tremendous impact on socio-economic development. Liver cancer is the cancer is one of the highest mortality rates in the world, the main pathogens of viral hepatitis, liver fibrosis and cirrhosis are liver cancer. Therefore, the diagnosis of liver disease has become an important part of medical diagnostics. The F protein of the human liver is one of the biological markers of a new not yet used in clinical direct reflection of liver damage extent. Beginning in the 1980s, people began to study the relationship between serum F protein content of the liver damage extent, some clinical data. The huge differences between the F protein concentration in the liver cells and serum F protein concentration as well as the F protein strict tissue specificity, and makes it a very promising reflect the sensitive and specific indicators of liver cell injury. Currently, the F protein of the liver-specific research and clinical applications by more and more attention. In this paper, on the basis of previous studies, the F protein expression plasmid pET15b-F was transformed into the expression strain BL21 (DE3) pLysS with IPTG to induce the expression of expression by affinity chromatography (Ni ^{2 < / sup>-chargedIDA His-bind column) purification, sodium dodecyl sulfate - polyacrylamide gel electrophoresis and immunoblotting (Western blot) to detect them. And protein immunized BALB-C mice to obtain spleen cells of the immune serum antibody titer was determined by indirect ELISA, select a high antibody titer mice were spleen cells with myeloma cells, preparation of single monoclonal antibodies. Human F protein expression strain BL21 (DE3) pLysS after the expression of whole bacterial proteins by SDS-PAGE detection, destination strip molecular size of approximately 43kD, is consistent with the literature reported. Western-blot shows the purified F protein and the guinea pig anti-human polyclonal antisera against the F protein was purified reaction is positive, indicating that by genetic engineering methods to obtain a purified F protein having immunological activity. The immune BALB-C mice were measured by ELISA serum antibody titers up to 1:72000, can be used for the preparation of monoclonal antibodies; culture supernatant of cell fusion the 15 holes antibody positive antibody titers up to 1:25600 Western-blot shows the antibody prepared by genetic engineering the expression of human liver-specific F protein the reaction, indicating that the antibody has the immunospecificity we prepared, can be used for subsequent large scale preparation of the monoclonal antibody and purification, as well as the development of immunological reagents. Liver-specific F protein reflect liver damage a sensitive, effective and stable indicators F protein in liver disease, early diagnosis and treatment, drug screening and Efficacy cure the situation, the judgment of the degree of liver injury and forensic medicine aspects, immune the tolerance liver transplant anti-rejection research has important application value. Chemical purification method is difficult to get a large number of F protein, which hindered the conduct of the study. Therefore, using genetic engineering methods to clone the F protein expression in human liver and induced the expression of human liver F protein to solve the problem of its origin. The same time this genetically engineered proteins as antigens preliminary preparation of monoclonal antibodies, and confirmed the specificity of this antibody by immunoblotting fumble of immunological reagents optimum conditions for the next step, immunological tolerance and immunological characteristics laid The basis for the clinical diagnosis of liver disease and treatment to open a new door.}

The Expression of the Liver Specific F Protein and the Preparation of Monoclonal Antibody

Abstract

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