Establishment Of HPV16E6 And HPV18E7 Oncoprotein Immunohistochemical Kit

Cervical cancer is the most common cancer which is caused by human papilloma virus(HPV)occurred in women,and has the second cancer death rate in women.In recent years,it has been found that the coverage of cervical cancer screening in rural areas of China is low,as a result,both the incidence and mortality rate of rural areas are higher than that in the city,showing a trend of rapid growth,and tend to be younger onset age,threat to women health seriously.Persistent infection of high risk HPV,especially HPV16 and HPV18 subtypes,is the underlying cause of cervical cancer.In addition,overexpression of E6 and E7 oncoproteins plays a critical role in the malignant transformation of cells,and are potential markers for early diagnosis of cervical cancer.Purposes:At present,there are no commercial immunohistochemical kits based on HPV16E6 and HPV18E7 used for detection of clinical samples.HPV DNA detection are the most widely used for clinical test,to the detriment of promotion in areas with scarce resources.To this end,it possesses superiority to establish immunohistochemical kits based on HPV16E6 and HPV18E7 oncoprotein with an independent intellectual property rights.Our aim is to establish HPV16E6 and HPV18E7 immunohistochemical kits,regarding HPV16E6 and HPV18E7 oncoprotein as diagnosis markers,with high specificity and low cost.And the kits serve as a tool for diagnosis and prognosis of cervical cancer.Methods:This paper used HPV16E6 and HPV18E7 recombinant proteins as antigen to prepare the monoclonal antibodies against HPV16E6 and HPV18E7,respectively.Then based on the monoclonal antibodies,the immunohistochemical kits for HPV16E6 or HPV18E7 oncoprotein detection in the cervical cancer cell lines was established.On the basis of the above,the immunohistochemical kit was used for the detection of clinical samples.Results:(1)Immuned the BALB/c mice with high-purity HPV16E6 and HPV18E7 recombinant protein.After subcloning screening,seven strains and eight strains of hybridoma cell strains that could stably secrete high-specificity and high-affinity antiHPV16E6 and anti-HPV18E7 monoclonal antibodies were obstained,respectively.And HPV16E6-4D4 and HPV18E7-E11 cell lines with the highest titer and belonged to IgG subtype were chosen to prepare and identify the monoclonal antibody;(2)Based on HPV16E6-4D4 and HPV18E7-E11 monoclonal antibodies,we established the HPV16E6 and HPV18E7 oncoprotein immunohistochemical kits in cervical cancer cell lines,respectively;(3)The HPV16E6 and HPV18E7 oncoprotein immunohistochemical kits were used to respectively detect clinical samples in different stages and have successfully detected the positive expression of HPV16E6 and HPV18E7 oncoprotein,and the coincidence rate of pathological examination results of cervical cancer was 95.23% and 100% respectively,showing good diagnostic performance.Conclusion:This study established HPV16E6 and HPV18E7 oncoprotein immunohistochemical detection kits,and used for the detection of cervical cancer and precancerous lesion clinical samples,showing the characteristics of low cost,better specificity,and can be used for early diagnosis of cervical cancer.