Construction And Identification Of A CDNA Library Of Human Gastric Mucosa

Gastric cancer is the most common gastrointestinal cancer in China, the annual prevalence and mortality rates are more than 2 times of the world average. The incidence of gastric cancer in China in a variety of malignant tumors in the first place, the second highest mortality rate. Since the first cases of gastric cancer surgery has been more than 100 years. From the present, surgery is still the primary means of gastric cancer.After more than a century of combined treatment of surgery on the development of gastric cancer survival rate has greatly improved. 5-year survival rate of first period gastric cancer is 82%-95%,of the second is 55%,the third is 15-30%,and the fourth is 2%. The problem is that there is still a high recurrence and metastasis. Neoadjuvant chemotherapy after surgery are used in advanced gastric cancer, but the effect is not obvious, and easy to produce drug resistance and apparent side effects.In recent years, gene diagnosis and treatment of cancer research has been a hot area of cancer research, it has become a cancer diagnosis and treatment of a new model. cDNA library can be constructedFor finding new genes and study gene function basis. Unlike genomic DNA which containing introns is difficult to express. Therefore we can use yeast two-hybrid method to filter to the desired gene from the cDNA library, and it be used for the purpose of gene expression. Therefore, building a cDNA library of gastric mucosal cells and further clonal selection, will help to understand the acute and chronic gastritis, gastric ulcer and digital gene expression profiles of benign and malignant tumors, screening with these specific diseases related gene and protein,then to investigate the acute and chronic gastritis, gastric ulcer and place the molecular pathogenesis of benign and malignant tumors and to find a basis for better treatment. And it will clarify to the development of gastric cancer from gastric cancer and the molecular pathogenesis of end-stage basis. Materials and methods: Use Trizol to extract total RNA from human normal gastric mucosa and use purified mRNA purification kit for mRNA purification. Then reverse transcription synthesis of single-strand cDNA.Use long-distance PCR to synthesis strand cDNA, after PCR products were proteinase K hydrolysis and purified, digested with XhoI. Fractionat of the digestion products,and recovery of the cDNA component which is more than 0.4kb. Then link with the pGADT7 vector. After the products is packaged in vitro protein, no amplified library is produced. Identify the titer and re-identification of library efficiency,and amplificat the library. Picked 16 plaques randomly, and use the vector cloning sites of universal primers for PCR amplification to test the quality of cDNA library.Results: The titer of cDNA library was 4.00×10~6 pfu/mL , the rate of recombinant was above 95 %, and the titer of amplified library was 9.50×10~9 pfu/mL . The insert size ranged from 0. 5 to 2 kb.Conclusion: The yeast two-hybrid cDNA library of human gastric mucosa was successfully constructed and can be used f or screening by Yeast Two-HyBrid to find the genes related to gastric diseases。...