Preparation And Identification Of Engrailed-2 Protein Monoclonal Antibody And Its Expression In Prostate Cancer

Objective:1.Expression of Engrailed-2 mRNA in human prostate cancer and benign prostatic hyperplasia2.Preparation and identification of Engrailed-2 protein monoclonal antibody3.Engrailed-2 protein expression in human prostate cancer and benign prostatic hyperplasiaMethod:20 cases of prostate cancer tissues and 25 cases of benign prostatic hyperplasia tissues were collected,and the expression of Engrailed-2 gene mRNA in human prostate cancer and benign prostatic hyperplasia tissue samples was detected using quantitative Real-Time PCR.The C-terminal 114aa sequence of the homeobox engrailed 2 gene（NM₀01427.3）was ligated into the gene expression vector pET-28a,transfected with DH5α E.coli,and expressed and purified EN2 recombinant protein.Mice were immunized with the EN2 recombinant protein,and hybridoma technology was used to screen cell lines expressing the EN2 monoclonal antibody.ELISA,Western Blotting and immunohistochemistry were used to identify the nature of the antibodies,and the positive cell lines were inoculated into the mice abdominal cavity mass production of EN2 monoclonal antibodies was performed.Subsequently,EN2 monoclonal antibodies were used to perform immunohistochemistry on human prostate cancer and benign prostatic hyperplasia tissue to analyze the expression difference of EN2 protein in prostate cancer tissue and benign prostatic hyperplasia tissue.Results:The mRNA expression of Engrailed-2 gene was higher in 20 human prostate cancer tissues than in the benign prostate hyperplasia group,and compared with the benign prostatic hyperplasia group,the expression of Engrailed-2 gene in prostate cancer was 1.45 times to 31.42 times.The pET-28a-EN2 recombinant expression vector was constructed successfully.After transfection into E.coli,an EN2 recombinant protein with a size of about 25 kD was induced.It immunized 4 BALB/c mice,of which 3 mice had higher serum titers,which could reach 1:3.2×104-1:6.4×104.After spleen cells of these 3 mice were fused with SP2/0 myeloma cells,after two rounds of ELISA screening,5B7,7E11,31D6,32E7,33H5,18H10,34E9,37C3 were finally screened.A total of 8 strains could produce EN2 monoclonal antibody hybridoma cell line.It was expanded and inoculated into the abdominal cavity of BALB/c mice,and the ascites was collected.The protein ascites was purified using Protein G affinity chromatography column,and finally 8 monoclonal antibodies against EN2 protein were obtained,which were 5B7（0.584mg/ml）,7E11（0.263mg/ml）,31D6（0.291mg/ml）,32E7（0.468mg/ml）,33H5（0.502mg/ml）,18H10（0.105 mg/ml）,34E9（1.831 mg/ml）,37C3（0.165 mg/ml）.After purification of 8 EN2 monoclonal antibodies,SDS-PAGE electrophoresis showed clear bands at 50kD and 25kD,corresponding to the heavy and light chains of the antibody,respectively.After titer determination,the antibody titers of 5B7 and 3 7C3 in the eight EN2 monoclonal antibodies were higher,and they could reach more than 1:1.28 × 104 for subsequent experiments.By Western Blot detection,the two EN2 monoclonal antibodies could specifically bind to the EN2 recombinant protein,and a clear band was seen near 25kD,which was consistent with the size of the recombinant EN2 protein.In addition,the two EN2 monoclonal antibodies can specifically bind to the EN2 protein in the total protein of the three prostate cancer cell lines PC3,DU 145,and LNCap.The development band is about 34kD,which corresponds to the size of the complete EN2 protein,and EN2 protein expression is higher in LNCap cell lines.The EN2 monoclonal antibody was used to perform immunohistochemistry in human prostate cancer and benign prostatic hyperplasia tissue,and it was found that EN2 was positively expressed in prostate cancer tissues,while EN2 protein was negative in prostatic hyperplasia tissues,and the expression of EN2 protein was also higher in tissues with high Engrailed-2 gene expression,such as issues 1 and 6.Conclusion:In the samples collected,the expression of EN2 gene in prostate cancer tissues was higher than that of benign prostate hyperplasia,ranging from 1.45 times to 31.42 times.Successfully prepared EN2 mouse monoclonal antibody.Experiments with self-made antibodies showed that the expression of EN2 protein was higher in LNCap cell line.The expression of EN2 protein in human prostate cancer tissue is significantly higher than that of prostate hyperplasia tissue.And the tissues with high EN2 gene expression have higher expression of EN2 protein.