| Antibody, which is employed extensively in diagnosis and treatment of illness as well as laboratory research, plays an important role in the field of modern medical science. The technology of Phage antibody library creates a whole new area for producing human monoclonal antibody. PCR was used to amplify from immunoglobulin all the variable fragments of heavy chain and light chain, which then was recombined into appropriate phagemid vector, and finally be displayed on the surface of phage by fusion with phage protein. Then, after panning, obtain the specific variable region gene without hybridoma or immunizing. In out study, we constructed a human phage antibody library by isolating lymphocytes from peripheral blood of normal human being, then cloned the variable region gene of heavy chain and light chain which was then recombined into express vector.1. Amplification of the variable region gene of antibody. This part includes following steps: ﹊solating of lymphocytes甧xtracting of RNA(3)purifying of mRNA畆everse transcription(aPCR. Rusult: the total quantity of extracted RNA is 1.05mg; purified mRNA is 11.97 u g. every variable region gene of VFK Vl^ VK has been amplified successfully using RT-PCR.2. The examination of efficency of digestion, ligation and electroporation which is very important for the purification level and capacity of library. Cutting the vector pDAN5 using different endozyme (Xho I > Nhe I > BssH II) in different buffer, and examining its cutting efficiency by means of electroporation; cutting a fragment from pDAN5,then putting it back after ligation to observe the ligation efficiency of fragment with vector; determination of efficiency of electroporation of pure plasmid and plasmid in ligation system. Results: Xho I x Nhe I > BssH II have a very high cutting efficiency while the cutting effiency of Sal I is only 50%. Xho I has a high cuttingefficiency not only in optimal buffer, but also in modified low salt buffer. Ligation of vector with fragment is 2-2.8 XI0s /jag vector. Therefore, it indicates that we can construct an antibody library with capacity of 109 6\I merely after ten to one hundred of electroporation, only if the two extreme point of fragment have been cutting perfectly by restriction endozyme; the electroporation efficiency of only-plasmid is 1 X 109~5X 109/ugDNA. But the electroporation efficiency of plasmid in ligation system is relatively low mainly caused by T4 DNA ligase. So. after heat inactive or extraction using hydroxybenzene , chloroform, this problem could be basically solved.3. The construction of T vector and its using in antibody library. The cloning of PCR product is a key step in construction of antibody library. But, the efficency of cloning of PCR product is very poor because of the insufficiently cutting of two points of PCR fragment due to the low cutting efficiency of PCR product. In this part, we try to find a way to solve this problem. The results indicate that: 畉he efficency of ligation of vector and fully cutting fragment, 1-1.4 X 107/ Ijil, is high enough for construction of antibody library. Therefore, obtaining the variable region gene fragment which has two fully cutting points is the key issue. (2)the difficulty of cutting of PCR product can not be easily solved by prolonging cutting time or increase the number of tails ? (3)similaly, the ligation efficiency of vector with PCR product which was cut directly. @ fortunately, the ligation efficiency of T vector with PCR product is quite high enough for the construction of antibody library, ﹎odifying the pSP73 to T vector.4. The construction of phage library of scFV antibody. First cloning variable region gene of light and heavy chain into Psp73-T respectively to get the light chain library and heavy chain library. Then, cloning the light chain fragement into pDANS after the light chain fragment was cutting out from Psp73-T. Using the same method, cloning the heavy chain fragement into the pDANS which has been inserted the light fragment previousl...
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