| Brain glioma is one of the common seen tumors in human, which is very harmful to their health, and the patients with glioma have a high mutilation and fatality rate. Although many therapeutic measures were employed, such as surgical operation, radiotherapy and chemotherapy, the patient's prognosis was still not satisfactory. Recently, immunotherapy has gradually become an investigation hot spot. It is said the method would become one of the most promising therapy. But nearly all antibodies used in the work were xenogenesis, which could not be applied to human body directly. So to produce new human antibodies against glioma has become an emergency problem. The phage displayantibody library as a revolution antibody engineering technology, which gets rid of the complex of hybridoma, provides a powerful means to produce human antibody. Now, we constructed a phage library expressing scFv antibodies against glioma, and in the meantimes performed a preliminary selection.1 .Construction of human scFv phage libraryPatients were assaied by immunohistochemistry method and ELISA. The positive of autologous antibody against human glioma was taken in. The peripheral blood samples of these patients were obtained. And lymphocytes were isolated from peripheral blood by Ficol solution. Total RNA were extracted, and cDNA were synthesized by RT-PCR. A set of oligonucleotide primers were designed and synthesized according to Kabat database. The variable region genes of human antibodies were amplified by nesting PCR, the products amplified in first cycle PCR were 700bp(VH> V K ); the second cycle products were 320bp(V K ) and 360bp(VH); the third cycle products were 400bp( VH ) and 450bp( V K ). Then the third cycle products were cloned into vectors pUC 18, VH and V K gene library were constructed respectively. Following ,VH were cloned into V K gene library7, and scFv gene library with 2X 107 capacity was constructed. Integrate scFv genes were obtained and cloned into display vectors pDNAS. These vectors were transfected E.Coli TGI, following primary labrary was constructed with the help of phage VCSM13. The E.Coli BS1365 were infected by phage of primary labrary so that scFv genes could be recombined in cell body. When E.Coli BS1365 were infected by VCSM13 helper phage, scFvs were displayed on the surface of phage. Then recombination library was constructed successfully. The titre of phage was 3.8X 1012. While E.Coli DH5 a F' were infected by the phageof recombination library and VCSM13 helper phage, the phage antibody library was constructed at last.2.Selection of phage antibodiesAntiboby library incubated with glioma cells, and pools of phage-scFv were absorbed to glioma cell line BT325. Bound phage were eluted at pH2.2 and infected E. Coli TG1. To minimize the binding of irrelevant phage antibodies and raise the rate of signal/noise, glioma cells were pre-incubate with phage library and diluted into an excess of normal glial cells. Glioma cells competed with glial cells for binding phage antibodies, then glioma cells were retrieved by anchorage culturing. And in order to obtain high affinity phage antibodies, a gradient elution was employed to eliminate those phage binding to cells at relatively low affinity.S.Cells binding ELISA assayAfter four rounds of panning, clones of the phage were tested by ELISA for binding to the glioma cells. About 40% of the clones tested showed positive reaction, and demonstrated the effectiveness of the panning procedure for selecting anti-glioma antiboies. These positive clones glial cells were tested for binding to the normal glial cells. 7 clones were selected as a result of negative reaction. After reappraisal of the 7 clones. 3 of them were identified as being specific which reacted with glioma cells but not with normal glial cells.
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