The Expression Of The Liver Specific F Protein And The Preparation Of Monoclonal Antibody

Liver disease may have a serious effect on our health,and will have a great effect on the social and economic development.Liver cancer is a malignant tumor which was one of the highest death rates in the world.Virus hepatitis,hepatic fibrosis, hepatocirrhosis and so on are the primary causes of liver cancer.So the diagnosis of liver disease is an important part of the medical diagnosis.The human liver F protein that is not applied to clinical medicine is a new kind of biological mark which can reflect the extent of the liver damage directly.Since the 1980s,people began to study the relationship between the serum concentration of F protein and the extent of the liver damage,and obtained some clinical data.It will be used for a very promising mark which can reflect the extent of the liver damage in high sensitivity and specificity by the great different concentration between the hepatocyte and serum,and also by the strict tissue specificity.Now medical study and application of liver specific F protein is being paid more and more attention.This study is based on the study of the predecessor.We obtained the expressing plasmid pET15b-F of F protein,and put this expressing plasmid into expressing strain BL21(DE3)pLysS and used IPTG to induce its expressing.Purification was done by affinity chromatograph(Ni²⁺-charged IDA His-bind column).The all stain protein after expressing was detected by SDS-PAGE and Western blot.Then we immuned BALB-C mouse with this protein to acquire immunity spleen cell,The indirect enzyme-linked immunosorbent assay was used to detect the titer of the antibody,the spleen cell which had higher titer antibody was selected to be fusioned with myeloma cell,so that we can prepare monoclonal antibody.The result of the expressing strain BL21(DE3)pLysS of human liver F protein was detected by SDS-PAGE,showing that the size of purpose protein was 43kD. This result was corresponded to the reports.The Western-blot showed that the strong immune reaction was occurred between purified protein and guinea pig antihuman extract F protein polyclonal antibody.This suggested that the purpose protein had the immunocompetence of human F protein.After immuning the BALB-C mouse,The indirect ELISA to be used for the titer of the antibody,the result suggested that the titer was hight to 1:72000,it could be used to prepare monoclonal antibody.After cell fusion,the culture supernatant had 15 wells positive antibody,the titer of the antibody was 1:25600.Western-blot suggested that the antibody prepared could be reacted with the extract human F protein,indicated that the antibody had the immunologic specificity,and it could be used for the continued study,such as large-scale preparation and purification of monoclonal antibody,the exploitation of immunoreagent.The liver specific F protein was a sensitive effective and stable mark to reflect the extent of the liver damage.And there was more important value in the early diagnosis and therapy about liver disease,drug screening and observation of curative effect,the condition of curing,judgement about extent of liver damage and forensic medicine,the study on liver transplantation antirejection of immunotolerance.It was so difficult to obtain a great quantity of F protein by chemical extract,this prevent the other studies' progress.So,we obtained the gene cloning of human liver F protein by gene engineering,and induced the expression of human liver F protein,solve the problem in source.And this protein was used to immunize the rabbit to obtain the monoclonal antibody,and the Western blot was used to detect the specificity.That provided the foundation for the next study on trying to find out the optimum condition of the immunoreagent,immunotolerance and immunological character.