Preparation Of LINE-1-ORF2 Antibody And Its Preliminary Application In Wilms Tumor Research

Background and ObjectiveWilms tumor(WT)is known as the most common malignant tumor in primary children urinary tract,accounting for 6% ~ 7% of children malignant tumors under the age of 15,of which about 75% in children is under the age of 5,and the onset of patients with congenital malformations is often before the age of 1,and the average age is about 3.5 years old.In recent years,with the comprehensive treatment program based on the individual-specific surgery,chemotherapy and radiotherapy combined with each other,it is possible to cure 85% of patients with WT.However,its individual-specific long-term treatment regimens are also accompanied by a higher prognostic risk at the same time,and a few children died of recurrence,metastasis,insensitivity to chemotherapy drugs or other factors.Complicated pathogenesis,many genetic and epigenetic factors involved,and lacking of targeted therapy drugs bring early diagnosis and treatment of great difficulties.A growing body of evidence shows that LINE-1 accounting for 17% of the human genome with potentially autonomous transposable activity is an important factor which influences the stability of the human genome.LINE-1 can make expression and transcription in human tumor cells,which shows that the transcription of LINE-1 may be in close connection with cancer.The phenomenon of the predominant low methylation of LINE-1 in tumors and the characteristic expression of L1-ORF1 and L1-ORF2 proteins in a variety of tumor cells indicate that LINE-1 has the potential to become a new tumor biomarker.LINE-1 low methylation phenomenon has been detected in the WT,but the relationship between the expression of L1-ORF2 and the pathogenesis of WT is still not clear recently.In view of the absence of L1-ORF2 antibody with good specificity and sensitivity at the same time,we propose to prepare L1-ORF2 antibody to detect the expression of L1-ORF2 in WT,and then to provide a new idea for studying the pathogenesis of WT and contribute to its early diagnosis and treatment.Methods In this study,L1-ORF2 p was prepared by gene engineering expression method.We immuned New Zealand white rabbit to make polyclonal antibody and detected its potency and specificity.And then immunofluorescence detection and overexpression of the WT cell line were studied by the antibody.Results 1.The recombinant plasmid p ET28a-L1-ORF2-EN was constructed and the L1-ORF2-EN fusion protein was expressed and purified.2.We use the purified L1-ORF2-EN fusion protein to immue New Zealand white rabbits to make antiserum.And then indirect ELISA,Western Blot and immunohistochemistry all showed that it has good titer and specificity and could be applied to the preliminary study of tumors.3.Immunofluorescence detection of WT-CLS1 cells(WT cells)by L1-ORF2 antiserum being a primary antibody showed that there was a distribution of L1-ORF2 in the nucleus and cytoplasm of WT-CLS1 cells.4.Make construction of the eukaryotic recombinant plasmid p EGFPN3-L1-ORF2 and transient transfection of WT-CLS1 cells.Western blot was used to detect WT-CLS1 cell lysates overexpressing L1-ORF2 proteins by L1-ORF2 antiserum.Then about 149 KDa specific strip was detected in it,with weaker strip of empty vecter transfected and untransfected ones,indicating that transfection can significantly improve the expression of L1-ORF2.Conclusions We prepared the L1-ORF2 protein by gene engineering expression method,and then immunized New Zealand white rabbits to prepare polyclonal antibody.ELISA,WB and immunohistochemistry were used to confirm that the prepared polyclonal antibody had good titer and specificity and could be applied to a preliminary study of tumors.We used the prepared polyclonal antibody todetect the expression of L1-ORF2 in WT cells by immunofluorescence and L1-ORF2 overexpression of the WT cell line,preparing for later research that the detection of the levels of L1-ORF2 proteins in WT cells under different medication,providing some references for the exploration of the development mechanism of WT,the choice of individual comprehensive treatment options and accurate treatments.