Construction Of Human Phage Single-chain Antibody Library (ScFv) For Gastric Carcinoma

BackgroundGastric cancer is a very common cancer whose incidence ranks the fourth among the common cancers. There are almost 934,000 new cases of gastric cancer occurring annually all over the world, among which about 0.7 million people die every year. In China, the mortality rate caused by gastric cancer is as high as 23.02%, ranking the top among all cancer deaths. At present, the main diagnostic methods of gastric cancer, like X ray and ultrasonographie endoscopique, are lack of sufficient accuracy and specificity for early diagnosis. Besides, the comprehensive treatments which are based on radiotherapy, chemotherapy and surgery suffer from a lot of restrictions in clinical applications because of too many side effects. Therefore, many researchers have shifted their focus onto the antibody diagnosis and treatment, which will be great helpful for the prevention and treatment of gastric cancer.The antibody technology has been widely applied to laboratory research, disease diagnosis and treatment and other areas and has demonstrated a significant role especially in disease prevention, diagnosis and treatment, making great contributions to the development of medicine. Phage antibody library technology which is based on PCR (Polymerase Chain Reaction) and PDT (Phage Display Techniques) provides an effective way of obtaining specific antibody molecules by constructing human-derived single-chain phage antibody library. This is not only a good way of obtaining humanized antibodies, but also an effective method of preparing antibody libraries which have higher capacity and better diversity. Furthermore, this technology has outstanding advantages in obtaining high affinity antibodies and reforming the antibody performance. This technology offers a simple but efficient operating system for preparing the humanized antibodies and shows its great significance in theoretical research and application onto the prevention, diagnosis and treatment of many diseases.Objective Construct human phage single-chain antibody library (ScFv) for gastric carcinoma by virtue of the new biological technologies:genetic engineering methods and phage antibody library technology and make a preliminary evaluation of the biological characteristics of ScFv so as to provide new sights for gastric cancer immunodiagnosis and immunotherapy.Methods1. Take the lymph node of primary gastric cancer patients as the source of B cells; Extract the total RNA of lymphocytes; Amplify the antibody heavy chain variable region (VH) and antibody light chain variable region (VL) gene fragments using RT-PCR technology; Digest and purify make use of gel extraction kit for the VH and VL gene fragments; Synthesize Linker chain and form VH-Linker-VL by connecting VL+Linker and VH. Finally, splice it into complete single-chain antibody (ScFv) gene fragments by introducing Sfi I and Not I at both ends of the ScFv fragments and using "overlap extension splicing polymerase chain reaction (SOE-PCR)" technology.2. Spliced between phage single-chain antibody library and phage vector pCANTAB-5E, constructed antibody library:Single-chain antibody ScFv gene fragments digested by restriction enzyme Sfi I and Not I with two steps respectively, reaction products were purified by agarose gel extraction kit, connected single-chain antibody ScFv and vector pCANTAB-5E through coupled reaction, the transformation efficiency attaining 6.0×107cfu/ug of competent E.coli TG1 were prepared, the phage vector pCANTAB-5E that inserted single-chain antibody ScFv were transformed into competent E.coli TG1, they super-infected by the helper phage M13KO7 subsequently, phage single-chain antibody library (ScFv) had been constructed through phage display, the storage capacity and recombination rate were further tested.Results1.The results show that the total RNA extracted from lymph nodes of gastric cancer patients have visible 28S and 18S bands in agarose gel electrophoresis, implying the integrity of RNA is good. In addition, it can be seen from the results that the VH gene fragment size is 375bp, the VL gene fragments size 325bp and ScFv gene fragment length 750bp after assembling.2. The 4.7×107cfu/ug resistant ampicillin clones were obtained from the experiment. Plating with appropriate bacilli bacteria had been transformed.10 clones were extracted randomly. The vector plasmids DNA were digested by SfiⅠand NotⅠrespectively after extracting, the result showed that the positive insertion rate was 80%(8/10). Then the sequencing results confirmed that antibody heavy chain and light chain insert the carrier accurately by means of the entire code of single-chain antibody manner and the homeology is 91%.ConclusionThe ScFv phage antibody library was successfully constructed in this experiment with a storage capacity of 4.7×107cfu/ug and the recombinant rate of 80%(8/10). The homeology of gene sequence between heavy chain and light chain variable regions is 91%. The results show that the fully humanized tumor-associated gene fragments can be obtained directly from the lymph node of the cancer patients through the phage antibody library technology, which provides a technological platform and insights for the preparation of a variety of antibodies in the future. It is sincerely hoped that the technology could provide technical and substantial supports for the sequential research on selecting specificity antibodies in the future.