Preparation And Functional Validation Of ZNF165 Monoclonal Antibody

Objective: ZNF165(Zinc finger protein 165)is a member of the oncotestin antigen family.Many studies have shown that it is highly expressed in various cancer tissues such as gastric,colorectal and esophageal cancers,as well as in testes and placenta,but not in other normal tissues.It promotes tumorigenesis and development through multiple pathways and is considered one of the important targets for immunotherapy.To address the current situation that there are no diagnostic and therapeutic reagents for ZNF165 antigen and the technical difficulties in its antibody development,this study successfully prepared ZNF165-specific monoclonal antibodies based on MBAT antibody development technology and further optimized the relevant technical points,aiming to provide a technical reserve for the establishment of new diagnostic methods and therapeutic antibodies for ZNF165 antigen,and to establish a technical system for the development of more monoclonal antibodies with potential clinical applications.Methods:(1)ZNF165 antigen expression plasmid construction and antigen preparation: ZNF165 full-length gene fragment was synthesized by GENE,and ZNF165 full-length and ZNF165(130-346aa)fragments were amplified by designing specific primers,and then the fragments were cloned into the enzymatically cleaved prokaryotic expression vector p ET-28 a.After the above constitutive plasmids were identified and sequenced,the full-length ZNF165 and ZNF165(130-346aa)recombinant proteins were induced by BL21 prokaryotic expression system,and the optimal expression conditions of ZNF165 full-length and ZNF165(130-346aa)recombinant proteins were explored by lysis,washing,dialysis and antigen purification of the inclusion bodies.We successfully obtained soluble ZNF165 recombinant protein and provided the basis for the next step of ZNF165-specific monoclonal antibody preparation;(2)Immunization of mice and determination of antibody potency: Balb/C mice were immunized with high purity and soluble ZNF165full-length recombinant protein according to the established protocol,and blood was collected from the tail vein at about 30 days of immunization,and antibody potency was measured by ELISA;(3)Establishment of positive B cell library and construction of ZNF165-Sc Fv plasmid library: After immunization,mouse spleen was taken and B cells were sorted,and positive B cell library was enriched by microfluidic technology.The positive B cell RNA was extracted and reverse transcribed to obtain c DNA,and then the positive B cell c DNA was amplified by multiple pairs of specific primers to obtain the ZNF165-Sc Fv sequence library;(4)Construction of 293 T cell ZNF165-Sc Fv antibody sequence display library and positive cell screening: the Sc Fv sequence was cloned into the Lenti-cmv-DP3 display vector,the above recombinant plasmid and 293 T cells were used to package the lentivirus,the 293 T cells were infected with the lentivirus,and a display library of 293 T cells expressing the specific ZNF165 antibody sequence was obtained through multiple screening by puromycin and microfluidic techniques;(5)Obtaining of the specific ZNF165-Sc Fv sequence and antibody screening and validation: extract the genome of positive 293 T cells after sorting,obtain the specific ZNF165-Sc Fv sequence after amplification,clone the specific antibody sequence into HZ002 expression vector,transfect 293 T cells to collect supernatant,screen and validate the antibody by ELISA and Western Blot.Results:(1)In this study,soluble ZNF165 full-length and ZNF165(130-346aa)recombinant proteins were successfully purified,but precipitation precipitated during the purification of ZNF165(130-346aa)recombinant protein,stray bands were present during the post-purification identification,and the concentration was low after quantification by BCA;(2)In order to ensure that highly specific antibodies were obtained,we used ZNF165 full-length recombinant protein as the immunogen to immunize mice and identified it by ELISA.The results showed that the antibody potency in mouse serum was high,which proved that ZNF165 full-length recombinant protein was immunogenic and the immunization protocol was reliable and reasonable;(3)Based on the MBAT antibody development technology,we optimized and improved the subsequent sorting and sequence screening methods,and obtained a ZNF165 monoclonal antibody with the best specificity by experimental verification,and obtained the unique Sc Fv sequence of this antibody by sequencing(the sequence is under patent application and not shown).Conclusion: We have successfully developed a monoclonal antibody specific for ZNF165 and obtained its Sc Fv antibody sequence;by optimizing the MBAT antibody development technology,highly specific monoclonal antibodies can be prepared in a shorter time and at a lower cost.Therefore,this research has effectively alleviated the lack of this monoclonal antibody,and the developed antibody is expected to be used for early antigen diagnosis and therapeutic antibody development for digestive tumors in the future.Secondly,this technology provides advanced technology reserve for the subsequent development of more monoclonal antibodies with new targets with clinical application potential.