Establishment And Preliminary Application Of The Method Of Fluorescence Immunotomography Of Liver Cancer-related Cytoskeleton Protein 4

Liver cancer is one of the most common malignant tumors in human beings.China is a country with a high incidence of liver cancer,and its morbidity and mortality rate are high.Since there are no obvious symptoms in the early stages,most patients are found to be in the middle and late stages with poor prognosis.Therefore,the early diagnosis of the disease is particularly important for the treatment and prognosis of the patient.At present,the commonly used serum diagnostic markers for liver cancer mainly include alpha-fetoprotein(AFP),prothrombin(PIVKA-?),and secreted protein Dickkopf-1(DKK1).These indicators are closely related to the occurrence and development of liver cancer It is related,but it has low efficacy in early liver cancer detection.Therefore,clinical application of new serum markers for early diagnosis of liver cancer is urgently needed to improve survival rate.In recent years,it has been found that cytoskeleton protein 4(CKAP4)is highly expressed in liver cancer tissue and has high sensitivity and specificity in the diagnosis of early liver cancer,which is expected to become an important indicator of high-risk liver cancer and early patient-assisted diagnosis.At present,there is a clinical shortage of CKAP4 markers of the bedside rapid detection method,therefore,this study intends to use fluorescent immunotomography technology,establish a serum CKAP4 fluorescence immunotomography quantitative rapid detection method,for high-risk groups of liver cancer and early patients to provide a simple operation,low-cost bedside rapid detection method.In this study,the strains containing CKAP4 gene recombinant plasmids were induced expression,optimized for induction temperature,expression time and inducer concentration respectively,The expression of the target protein was detected by SDS-PAGE and Western Blot,and the target protein was purified by affinity chromatography to obtain CKAP4 antigen.According to the reference preparation standard,repeatedly calibrate the CKAP4 antigen to prepare the indoor reference,gradient dilution to prepare the CKAP4 fluorescent immunochromatography precision,linear reference,and at the same time to explore the stability of thereference.In the establishment of CKAP4 protein time-resolution fluorescent immunomomomography detection method: We respectively from fiberglass cotton,nitric cellulose membrane,antibodies and other aspects of raw materials inquiry,from the labeling antibody volume,the concentration of packaged antibodies,activation agent dosage,buffer p H,protective liquid group and other aspects of the process and group research.The test paper was prepared by the optimized process,and the performance evaluation of the test paper was carried out from the aspects of linearity,precision,sensitivity,accuracy,specificity and stability.This study determined that the best induction conditions for BL21(DE3)/p ET32a-CKAP4 recombinant bacteria: induction temperature was 37 degrees C,induction time was 4 h,IPTG concentration was 1.0 mmol/L.Through SDS-PAGE electrophoresis detection,the target protein is expressed in the form of inclusion,and the molecular weight is consistent with expectation,the protein antigen is good through Western Blot,and the protein was effectively renatured by gradient dialysis.Using the renatured target protein,prepare an indoor reference product with CKAP4 protein content of 20 ?g / m L and 50 ?g / m L,respectively.The prepared reference product can be stored stably at 4 ? for 1 week,and 12 at-80 ? month.A rapid detection method of CKAP4 fluorescence immunotomography was established.The linear detection range of CKAP4 is: 25-1000 pg/m L,the minimum detection limit is 15 pg/m L,the coefficient of variation in batches and batches is within 15%,in line with the precision requirements,and the recovery rate is between 85%-115%,with the liver cancer marker AFP,PIVKA-II,no cross-reaction.It can be stored for 20 days at 37 degrees C,the stability is good,and by testing 87 samples,the results show good correlation with ELISA.In this study,CKAP4 was successfully expressed in the primary nuclear expression system,c KAP4 recombinant protein antigen with immunological activity was obtained,which can be used as an immunogen and indoor reference product preparation,and a set of linear and precision indoor reference product can be used for CKAP4 fluorescence immunotomography test paper detection.A rapid detection method of CKAP4 fluorescence immunotomography was established to provide a fast,sensitive,simple,economical and single-handed testing method for high-risk groupsof liver cancer and early patients.