Isolation Of Single-chain Variable Fragments Against Human CXCR4 By Yeast Two-hybrid System And Their Preliminary Analysis

Purposes: Chemokine receptor 4(CXCR4) is a transmembrane glycoprotein(40 kDa) and is considered as a cancer stem cell surface marker. In recent years, many studies have shown that CXCR4 plays an important role in tumor metastasis and invasion. In this study, the single-chain variable fragments(scFVs) against CXCR4 were screened by yeast two-hybrid technique. sc FVs can be further studied for their anti-cancer activities in the future.Methods: The recombinant bait vector pGBKT7-CXCR4 was transformed into yeast strain AH109 and tested for self-activation in nutrient-deficient medium. The sc FVs library was transformed into AH109, which contains the bait gene, and was screened in SD/-Trp/-leu/-His+10mM 3-AT medium. The identified clones were checked in SD/-Trp/-leu/-His/-Ade +10mM3-AT medium. The positive clones were further transformed into AH109 containing empty vector pGBKT7, bait vector pGBKT7-CXCR4 or unrelated antigens vector as negative controls, respectively. The positive clones were sequenced, and their DNA sequences were analyzed. The positive clones were inserted into the expression vector pET-28a-sumo, and the expressed proteins were purified as the form of inclusion bodies. ELISA was performed to examine the binding of these purified proteins with the second extracellular loop of human CXCR4.Results:(1) The recombinant bait vector pGBKT7-CXCR4 was constructed and did not show self-activation.(2) In this study, a total of 3 different scFVs against CXCR4 were identified by the yeast two-hybrid system. The DNA sequences analysis showed that those 3 clones were scFVs and named as aCX73, aCX82, aCX136.(3) These 3 scFVs clones were inserted into the expression vector pET-28a-sumo, and two of the expressed proteins(aCX73, aCX82) were successfully purified as inclusion bodies.(4) The ELISA analysis showed that aCX73 and aCX82 could strongly bind to human CXCR4. These scFVs can be further studied for their anti-cancer activities.Conclusions: After the human sc FVs library was screened by yeast two-hybrid technology. Three sc FVs(aCX73, aCX82, aCX136) were isolated against human CXCR4. Two of the scFVs(aCX73, aCX82) were purified as inclusion bodies and showed a strong binding to human CXCR4. This study laid a good foundation for the further study of these sc FVs for their anti-cancer activity in vitro and in vivo.