| Antibody library displayed on phage surface was a new technique developed in the last ten years. But because the repertoire is too small, it's use was limited to selection antibodies of the known antigens. Another restriction was that the material of the libraries are mainly the spleen of mice. For all these reasons, it couldn't be applied widely. It was the reason that most people were despond to it after they have being excited when it appeared at the beginning. Later, with the techniques improving, large library (> 108)was not a challenge that could not be tapped any more. For another time, people took a fresh look at it, of it's developing and it's application. One advantage of the large library was that a naTve library could be constructed without immunization which became a new method to prepare human antibodies. Another advantage was that it was possible to select antibodies to unknown antigens, for example, tumor specific antigens. Especially, after the recombination system in vivo was applied to antibody library construction and the repertoire reached to a striking level that couldn't be taped before, the two advantages became more remarkable. Although it sounded charming, there were still problems remained. One of these was the library contamination, for only 1/4 of the library is the rightly assembled scFv gene. The other 3/4 useless library would bring unexpected trouble on the next step panning. Bradbury do more improvements on the in vivo recombination technique, which not only obviated the library contamination, but also ensured that the diversitv of the library couldn't be lost. Anotheradvantage of the recombination of Bradbury was that shuffling was performed before every panning, which made it possible that antibodies with higher affinity would be obtained.We adopted the loxp-Cre system used by Bradbury that recombination could be performed in vivo to construct a 8X10U scFv library of HCC. The methods and results are as follows: the lymphocytes were isolated from 400ml peripheral blood donated by about 200 HCC patients. Then Img total RNA was isolated from these lymphocytes and 12 u g mRNA was further obtained. RT-PCR were performed using the primers annealing to the first and fourth frame domain of the V region. The PCR products were mixed and the second PCR was performed with the primer annealing sites being the common tails added to the 5' end of the primers of the first PCR. The products of the second PCR were cloned into T vector. So the cloning library of VH and VL were constructed. The repertoire of VH was 3.85><108and the VL 2.30*107. The scFv library construction was accomplished in a stepwise way. That was the VL fragments being cloned into pDANS first and followed by the cloning of the VH fragments. Phage rescue was performed with the helper phage M13K07 and the primary library was obtained. Then the BS1365 was infected by the primary library so the VH and the VL fragments were shuffling in the BS 1365 bacteria. After the phage rescue the secondary library with the repertoire of 8*10" were obtained.Different from the ordinary gene clone, library' construction requires high efficiency of every process. For this reason, a seriespre-experiments would be performed to optimize the conditions for every process. This mainly includes the digestion efficiencies of the vector by restriction enzymes, the cloning efficiency of the PCR products and the transformation efficiency. These processes are the restriction steps that determine in a large degree that a large library could be constructed or not. The low digestion efficiency of the PCR products brought a disadvantage result that the cloning efficiency of the PCR products directly into the phagemid was very low. To resolve this problem, we cloned the VH and VL genes into T vector first, then cut them out and cloned into the phagemid in a stepwise way. This alteration of the traditional library construction procedure raised the cloning efficiency of PCR products more than 20 folds, which made the construction of a large libra...
|
PDF Full Text Request