Preparation Of Novel DsCystatin Nanobody And Studies On The Mechanism Of Methylation-regulated USP44 Gene In ESCC

There’s a kind antibody without a light chain expressed in the camel serum,by cloning its variable region,an antigen-binding fragment with the smallest known molecular weight named nanobody is obtained.Compared with traditional antibodies,nanobodies have low molecular weight,high hydrophilicity,and are easily covalently linked to other molecules or prodrugs.Therefore,it has broad prospects in terms of new drug development,clinical diagnosis,and so on.The Cathepsin protein family participates in antigen degradation,processing,and delivery within immune cells,thereby regulating the host immune response.When the human body is bitten,the Ds Cystatin protein in the tick’s salivary glands is secreted into the body,inhibiting the activity of the Cathepsin protein family,helping the tick to suck blood on the host for a long time,evading the host immune response,and promoting the spread of pathogens carried by ticks.We hope to obtain Nanobodies against Ds Cystatin protein,which can play roles in the diagnosis and treatment in bites.In this study we used a prokaryotic expression system to obtain the GST-tagged Ds Cystatin fusion protein,which was purified using a GST tag.Then we excised the GST-tag using a procession protease to obtain a single Ds Cystatin antigen protein.We immuned healthy Bactrian camel several time using the purified Ds Cystatin antigen protein,then established a nanobody immune library using its peripheral blood as a material.We utilized phage display technology,by absorbing,eluting,and enriching to screen Nanobodies with antigen-specificity,ELISA to obtain positive monoclonal antibodies with specific binding to Ds Cystatin protein.The positive monoclone in the ELISA experiment were amplificated,and plasmid extraction and sequencing,finally 1 nanobody was obtained.We used the pull-down technique to verify that our Nanobodies interact with the Ds Cystatin protein.Based on another research direction of the laboratory,we also studied the biological mechanisms of USP44 which is regulated by methylation in esophageal squamous cell carcinoma(ESCC).In this study,we applied statistical principles,analysed differential sites of methylation in ESCC in TCGA database,combined with the sequencing results of methylation differences in 94 pairs of esophageal cancer tissues in our laboratory to finally determine that USP44 presented a significant abnormal hypermethylation.We used logistic regression to calculate the diagnostic efficacy of this gene for ESCC.The results showed that the diagnostic sensitivity was 0.73,the specificity was 0.83,ROC area under the curve(AUC)is 0.82,meaning that USP44 has clinical diagnostic value of ESCC.To further explore the biological mechanisms of the methylation-regulated gene USP44,we used demethylating drugs(5-Aza)to treat the esophageal carcinoma cell lines Ec-109 and Ca Es-17 and found that the expression of USP44 increased significantly.Then we constructed a luciferase expression vector targeting the promoter region of USP44 gene and hypermethylated the promoter region,we found that the fluorescence value was significantly lower than negative control.In patients cancerous and paracancerous tissues,the expression of USP44 gene also showed a significant difference.These results suggest that the expression of the USP44 gene in ESCC is regulated by DNA methylation,and hypermethylation leads to low expression of this gene.To further explore the mechanism of USP44 in ESCC,we constructed the p CDH-USP44 overexpression vector.After cell proliferation,and cell migration experiments,the gene was shown to inhibit tumors in ESCC,and apoptosis experiments also showed that the gene can promote apoptosis of ESCC cell.Finally,in order to assess the role of the USP44 overexpression in tumor formation in vivo,we performed a tumor-bearing test in mice.According to the statistical results of tumor volume,it was demonstrated that the USP44 gene plays a role in suppressing tumor size in vivo.