Expression Of Pregnangcy Associated Plasma Protein A Based On Epitope Analysis And Study On Its Clinical Application

Objective:We prepared recombinant pregnancy-associated plasma protein A (PAPP-A) based on the analysis of epitopes of the CDS region and the protein was injected into two rabbits to make PAPP-A polyclonal antibody, then we prepared HRP and biotin labeled antibody. Then the BA-ELISA kit was made to detect PAPP-A concentrations in serum of patients of DS or ACS, the aim was to detect the associated diseases, which was meaningful for therapy.Methods:1. The representative epitopes of CDS region of PAPP-A were analyzed using the software of DNAstar.2. Two placentas were collected and used to abstract total RNA, then the DNA was made after PCR, we used plasmid of PAPP-A transformed bacteria, which was cultured and made DNA.3. The obtained PAPP-A DNA was connected to the plasmid of pET42a after restriction, the transformed into the bacteria of TOP10. After identified, it was transformed into the bacteria of BL21, so the recombinant PAPP-A protein was prepared. The protein was purified by nickel column.4. PAPP-A antibody was made by injecting the recombinant PAPP-A protein into rabbits. PAPP-A antibody was purified by SPA column.5. PAPP-A antigen in serum of pregnant women was purified by immunity chromatography, ion exchange chromatography and molecular sieve chromatography.6. We made HRP and biotin labeled PAPP-A antibody and optimized reaction conditions.7. The sensitivity, repeatability and stability of the BA-ELISA method were tested.8. We detected the samples of DS and ACS to find the clinical applications.Results:1. We used the software of DNAstar and compared the analysis of signal peptide, the 1to309bp were picked out for it has signal peptide, which made the protein could pass through membrane.2. DNA of PAPP-A transformed into bacteria was obtained, and we managed to get the DNA from expand cultured bacteria.3. Recombinant PAPP-A protein was successfully prepared according to the usual method.4. Two New Zealand rabbits were injected with the prepared PAPP-A antigen to get PAPP-A antibody, and the titer was tested using the method of double agar diffusion, it was about1:30.5. The column was added with the antibody connected with the gel.6. NaI4was used to make the HRP labeled antibody, the enzyme we got was what we want. The concentration was detected, which was1.2mg/mL. Biotin labeled antibody was also successfully prepared.7. Serum samples of full-term pregnant women were purified with affinity chromatography, ion exchange chromatography and molecular sieve chromatography. The PAPP-A was detected with SDS-PAGE, and a single band in the gel.8. BA-ELISA method for detecting PAPP-A in serum of different trimesters has significant value in clinical application. So was the method established to detect PAPP-A in patients of ACS.Conclusions:This study prepared the recombinant PAPP-A protein in view of the epitopes, avoiding interruptions of others and made it be easy to make the protein. Antibody could be made by injecting rabbits and purified by chromatographies, then connected with HRP and biotin to establish BA-ELISA system.Methodology indexes of the method were all good. The method was fit for detecting situations of fetus of different trimesters and patients of ACS. BA-ELISA method provided the tool to detect PAPP-A rapidly, precisely and conveniently, and made it had significant value in clinical application.