| Human serum albumin, produced by liver, is the most protein in human blood and maintains the dynamic equilibrium between blood and tissues. HSA, a drug in forms of injection including liquid preparations and lyophilized powder, isolated and purified by the cold ethanol way from the safety blood and virus inactivated, has been called lifesaving drug since it's often used in many clinical diseases. HSA is always in shortage in recent years because its raw material, i.e. human blood, is short of supply for its production. The imbalance between its limited supply and huge clinical demand results in various fake HSA medicines and even criminals who seek for the huge illegal profits. The counterfeits threatened not only HSA itself, but also patients ? security, which is indeed an undeniable and unforgiving fact. And also, the forms of fake HSA have had the trend of shifting from chemical composition to different species serum albumin. However, an exclusive, effective and fast method for identification of fake HSA has not been developed till now. The objective of this thesis is to establish a fast, effective and pivotal method for distinguishing HSA from other fake HSA. On the basis of immunodominant epitope screening, using molecular biology techniques, an artificial antigen and artificial antibody were constructed in this study and a fast pivotal novel method for HSA identification has been established for the first time.HSA gene sequence was obtained firstly and the Biosun software was used to predict the B cell antigen epitope of human, horse, cow and goat serum albumin coding sequence, respectively. At the same time, the difference between these sequences was compared by ClustalX. By the predicted results, the three special amino acid sequence, that is, 126-162 aa, 314-355 aa and 373-424 aa, were cloned and expressed, respectively. Based on central template, the designed gene sequence primers were synthesize by PCR firstly, then combined with the carrier PGEX-4T-2 and inserted into E.Coli HB101. The antigenicity of objective proteins were tested by indirect ELISA by HSA antibody and its specificity was tested by reacting with horse, cow, goat serum albumin antibody, respectively. The specific antigens and HSA were taken to immune the rabbits and then serum antibody were obtained and purified by octanoic acid-ammonium sulfate, evaluated by IC50 and Kids. Finally, by using HSA monoclonal antibody as coating antibody and the polyclonal antibody as the second antibody, we build a double antibodies system to detect the HSA.Target fragment production by molecular clone was recycled and enzyme cleavage production was then evaluated by agarose gel electrophoresis. And there are target strips around the 110, 130 and 150 bp fields. The target fragments linked to the carriers and then expressed in E.Coli HB101, evaluated by SDS-PAGE. There were target strips around the 30.1, 30.6, 31.7 kD fields. In the experiment, the 373-424 aa was found as the immunodominant epitope of HSA and the corresponding antibody compared with HSA antibody. The IC50 were 1.635?g·mL-1, 3.280?g·mL-1 and the Kids were 2.438×10-11, 4.211×10-10, respectively. The results showed that the antibody obtained in the study is specific and have elective affinity, and the detection system is effective to identify HSA.In brief, we obtain the immunodominant epitope sequence, the corresponding antigen protein and monoclonal antibody by prokaryotic expression and in vitro immunization technology. The most significant aspect of the thesis, a rapid pivotal novel ELISA detective method for HSA identification has been established for the first time. |
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