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    Master's thesis | Liu Jing of Jinan University used yeast two hybrid technology to screen single chain antibody against human CXCR4 and its preliminary analysis


    Tumor is a common and frequently occurring disease. Currently, malignant tumors are the most serious threat to human health. For the treatment of malignant tumors, radiotherapy, chemotherapy, drug assisted therapy, and biological therapy are generally used. However, due to the proliferation, metastasis, and recurrence of malignant tumor cells, the treatment effect and prognosis are poor.

     

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    CXCR4, as a surface marker of tumor stem cells, is closely related to the malignant development of tumors. Secondly, single chain antibodies have a relatively small molecular weight, are easy to penetrate into tumor tissue, have low immunogenicity, short half-life, and can target tumor tissue alone or in combination with certain drugs to reduce damage to normal tissue. This is also a hot topic in antibody engineering, coupled with the advantages of yeast two hybrid technology. Therefore, CXCR4 was selected as the target and yeast two hybrid technology was used to screen single chain antibodies against CXCR4 from human single chain antibody libraries, providing a foundation for the development of antibody drugs for the treatment of malignant tumors.

     

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    Protein protein interaction exists in the process of life activities of body cells and is an important basis for life activities. Traditionally, protein immunoimprint, immunoprecipitation and other methods are used to detect protein interactions, but these methods are only used to detect in vitro, and can not simulate the situation of protein interactions in human body. In 1989, Fieds and Songs first proposed and established a new genetic method for directly detecting protein interactions within cells, known as the yeast two hybrid system. Subsequently, the system continued to improve. Yeast two hybrid technology not only detected protein-protein interactions, but also detected proteins with unknown functions. Even weak and instantaneous connections between proteins, antigens, and antibodies could be sensitively detected based on the reported gene expression products, playing a very important role in protein research.

     

    Therefore, we first constructed a CXCR4 bait fusion protein expression vector and screened single chain antibodies interacting with CXCR4 using yeast two hybrid technology. The screened single chain antibodies were purified and further analyzed for their anti-tumor activity. Due to the possibility that some proteins may have activated reporter gene expression when transformed into yeast strain AH109, self activation detection of bait proteins is necessary before library screening. In this experiment, p GBKT7-CXCR4 was transformed into AH109 to verify its self activation effect. p GBKT7 carries Trp as a nutritional marker in yeast strains and can therefore grow on the - Trp plate. In yeast AH109 containing pGBKT7-CXCR4, the DNA-binding domain (DNA-BD) can bind to the upstream activation sequence (UAS), but lacks the binding of the transcriptional activation domain (DNA-AD). Therefore, the reporter genes ADE and HIS3 regulated by the downstream promoter of GAL4 UAS cannot be expressed by transcriptional activation, that is, they cannot grow on the culture of SD/- Trp/- His, SD/- Trp/- Ade. The experimental results in Figure 3-3 indicate that yeast AH109 containing bait protein does not grow on SD/- Trp/- His, SD/- Trp/- Ade cultures, indicating that the bait protein does not have self activation. Therefore, the p GBKT7-CXCR4 plasmid is suitable for screening single chain antibodies in yeast two hybrid systems.

     

    The yeast two hybrid system used in this experiment is GAL4 Two Hybrid System3, and yeast strain AH109 was selected. The strain has already knocked out the GAL4 gene and cannot express GAL4 endogenous protein. The four reporter genes HIS3, Ade, MELI, and Lac Z regulated by GAL4, including His and Ade, are used for nutritional screening. In this system, there are two different plasmids, including the bait plasmid with Trp nutrient marker and the library plasmid with Leu nutrient marker. His and Ade reporter genes can provide strong nutritional screening, while 3-AT can inhibit His background expression, increasing the rigor of screening. The screened monoclonal clones were lined on SD/- Trp/- leu/- Hiss/- Ade+10m M3-AT plates for further identification of candidate positive clones.

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