Establishment Of Fluorescence Immunochromatography Combined Detection Of HPV16/18 E6 Protein

BackgroundCervical cancer is the most common malignancy in women after the incidence of breast cancer.High-risk human papillomavirus(HPV)persistent infection is the leading cause of cervical cancer and precancerous lesions,and more than 80% of cervical cancers are associated with high-risk HPV types 16 and 18.When the patient is persistently infected with the high-risk HPV 16/18 type,E6 protein is synthesized in a large amount in the cervical epithelial cells,and the malignant transformation of the cervical epithelial cells is caused by inhibiting the activity of the tumor suppressor P53,activating telomerase,and finally leading to cervical precancerous lesions and cervical cancer.Therefore,the detection of HPV16/18 E6 protein in cervical epithelial exfoliated cells can provide evidence for the early screening and auxiliary diagnosis of cervical cancer and precancerous lesions.However,due to the use of cervical epithelial exfoliated cells as a test sample,it is greatly affected by the sampling error,and there is a lack of clinical methods for rapid quantitative detection.ObjectiveTo establish a quantitative and rapid method for the detection of HPV16/18 type E6 protein in human cervical epithelial cells with housekeeping protein ?-Actin as internal reference,and to provide a sensitive and specific method for early screening and auxiliary diagnosis of cervical cancer and precancerous lesions.Simple and low cost detection method.Methods1.Recombinant expression and purification of HPV18 E6 protein: The p ET28a-HPV18E6 plasmid was transferred into BL21DE3 strain.The concentration of inducer,induction time and induction temperature were optimized by single factor variable method,and the target protein was expressed.The protein was purified by Ni affinity chromatography.2.Reference product development: According to CNAS-GL29:2010,a standard product preservation solution was used to dilute HPV16/18 E6 protein antigen and ?-Actin protein development laboratory reference product.3.Development of HPV16/18E6 protein fluorescent immunochromatographic assay test strip: the anti-?-Actin monoclonal antibody,anti-HPV16 E6 and HPV18 E6 monoclonal antibodies were labeled with fluorescent microspheres.On the same NC membrane,HPV16 E6 paired monoclonal antibody was used as the detection line T1,HPV18 E6 paired monoclonal antibody was used as the detection line T2,and ?-Actin paired monoclonal antibody was used as the internal reference line A,and the test strip was prepared using the principle of fluorescence immunochromatography and the double antibody sandwich method.4.The test strip process optimization and performance evaluation: Optimize the main raw materials and processes for the development of test strips,and thro?gh the detection of linear range,precision,accuracy,stability,and clinical trials for performance evaluation.Results1.When the recombinant strain was induced at 37? and 1.0 mmol / L IPTG for 4 h,the recombinant HPV18 E6 protein with molecular weight of 18 KDa was expressed in large quantities,which was proved to be immunogenicity by Western blot,and the purity of the recombinant protein was 95% after affinity purification.2.A total of 45 references including linear range,precision and other performance evaluation materials were prepared.3.A rapid quantitative fluorescent immunochromatographic assay for the detection of human papillomavirus 16/18 E6 protein in cervical epithelial cells with housekeeping protein ?-Actin as internal reference was established.The linear range of HPV16/18E6 protein were 2.0-200 ng/ml;the average recoveries were 102.4% and 98.1%,respectively.There was no significant cross reaction with squamous cell carcinoma antigen(SCCa)and human epididymis protein 4(HE4).The intra-batch CVs were all less than 10%,and the inter-batch CVs were all less than 15%.Clinical experiments were carried out to detect 46 samples of cervical cancer patients,38 samples of CIN III patients with precancerous lesions,who diagnosed by clinical pathology and 52 parts of HPV DNA negative samples.The results showed that the sensitivity of screening for cervical cancer and precancerous lesions was 91.3%,84.2%,and specificity of 100%.Conclusions1.The recombinant HPV18 E6 protein with 95% purity was obtained.2.A set of indoor reference products for the combined detection of HPV16/18E6 protein fluorescence immunochromatographic test strips was prepared.3.A rapid quantitative method for the detection of human papillomavirus type 16 / 18 E6 protein in cervical epithelial exfoliated cells with housekeeping protein?-Actin as internal reference was established,which provides a high sensitivity,good specificity and low cost detection method for early screening and auxiliary diagnosis of cervical cancer and precancerous lesions.