Preparation Of Lung Cancer-related IL1R1,Galectin-3 And MUC1 Protein And Detection Of Their Sandwich Immunosensors

Lung cancer has always been the most common cancer in the world,after breast cancer and prostate cancer,the third most common cancer,with the largest proportion of cancer deaths and poor prognosis.Early lung cancer generally does Not produce obvious symptoms,about 60%of people are already in the advanced stage of the disease at the time of diagnosis and can Not receive surgical treatment.The 5-year survival rate of patients is less than 15%.If lung cancer can be detected early and surgical resection is performed in time,the postoperative mortality rate of patients can be reduced to 2%.Therefore,early diagnosis plays a vital role in improving the survival rate and prognosis of lung cancer patients.The traditional diagnostic methods for lung cancer mainly include imaging examination,bronchoscopy,and pathological biopsy.Among them,the imaging diagnosis is difficult to differentiate between benign and malignant nodules.Pathological biopsy needs to consider the patient’s physical condition,and also has certain risks.Therefore,early diagnosis and prediction of cancer markers have become a research hotspot.Serum tumor markers are biological indicators detected from the serum or plasma of patients with suspected tumors.These indicators can reflect the existence of tumors,which is conducive to the pathological analysis and development evaluation of tumors.Compared with image examination and pathological examination,tumor marker detection is convenient,the cost of the instrument is low,and the invasion is few.Therefore,this subject studies the combined detection of IL1R1,Galectin-3,and MUC1 protein expression in the serum of lung cancer patients by the current-type sandwich immunosensor.The method has the advantages of low cost,simple preparation,convenient operation,small sample size,fast,one-time,To provide technical support for the early detection of lung cancer patients.The main results are as follows:1.Select the Galectin gene（Galectin-3）,migratory and invasive interleukin-1receptor protein 1 gene（IL1R1）and MUC1 mucin gene（MUC1）related to lung cancer,find their CDS sequence,and select specific gene fragments,By PCR amplification,the IL1R1 gene fragment was 486bp long,and the Galectin-3 gene fragment was 753bp,and MUC1 gene fragment length of 513bp.The prokaryotic expression vectors of these gene fragments were successfully constructed,transformed into E.coli-derived strain Rosetta,These three soluble recombinant proteins were obtained by protein expression purifications.Determination of the concentration of purified recombinant protein by Brand Ford method,The measured IL1R1,Galectin-3 and MUC1 protein concentrations were 10.5μg/m L,50μg/m L and 27μg/m L,respectively.2.Preparation of specific antibodies:The obtained recombinant proteins IL1R1,Galectin-3 and MUC1 were fully ground with Freund’s adjuvant to form a qualified oil-in-fill agent.Immunize rabbits.,After immunization four times,collect blood from rabbit heart to obtain corresponding specific antibodies.Western Blot method showed that IL1R1,Galectin-3,MUC1 antiserum can specifically bind to its corresponding antigen,And the use of agarose two-way immunodiffusion method for IL1R1,Galectin-3,MUC1 antibody titer analysis.The antibody titers of IL1R1,Galectin-3and MUC1 were initially obtained as 1:16,1:32 and 1:32,respectively.3.Preparation of current-type sandwich immunosensor:Choose carbon paste as the working electrode,silver chloride as the reference electrode,and carbon paste as the counter electrode.Assemble the multi-walled carbon na Notubes/gold nanoparticles（Mw CNTs/Au NPs）and the detection protein antibody in turn on the working electrode of the carbon paste printed electrode to prepare the SPCE│Mw CNTs-Au-detection protein antibody electrode,The non-specific binding site was blocked with bovine serum protein BSA.The detection protein capture antibody probe is prepared,Add antibody protein and catalase（CAT）to the colloidal gold solution to obtain the signal antibody probe of CAT nanogold labeled detection protein.After the capture antibody probe is physically adsorbed to the working electrode,After adding the detection protein sample,due to the detection protein antibody specifically binds to desmin,the carbon electrode can quickly identify and capture the detection protein in the sample,and then add a detection protein signal antibody probe.The detection protein antibody on the probe can quickly identify the detection protein captured by the carbon electrode,therefore a current-type sandwich immunosensor was fabricated.The CAT enzyme on the signal antibody probe catalyzes the addition of H₂O₂to produce a reductive oxidation reaction,and the signal current peak of the reaction is detected by an electrochemical analysis workstation,which can quickly detect the expression level of the detected protein.Current-type sandwich immunosensors for IL1R1,Galectin-3 and MUC1 protein detection were prepared by the above methods.4.Current-type sandwich immunosensor detection of peripheral blood tumor markers in lung cancer patients:Serum samples and pathological data of 25 lung cancer patients were collected from the First People’s Hospital of Kunming City.The electrochemical sandwich immunosensor and Western Blot were used to detect the expression of IL1R1,Galectin-3 and MUC1 protein in the serum of lung cancer patients.The Western Blot method can detect these three proteins in 25 blood samples.The concentrations of IL1R1,Galectin-3 and MUC1 protein were measured in6.28-11.66 ng/m L,1.04-2.36 ng/m L and 1.31-2.64 ng/m L.The current-type sandwich immunosensor can also detect these three proteins in 25 blood samples.The measured protein concentrations of IL1R1,Galectin-3 and MUC1 are 7.01-12.36 ng/m L,1.11-2.50 ng/m L and 1.65-2.79 ng/m L,respectively.The protein concentration detected by the same sample sensor is about higher than that detected by Western Blot method.The concentrations detected by the two methods were significantly positively correlated.The correlation coefficients of Person detection IL1R1,Galectin-3 and MUC1 were divided into 0.672,0.913 and 0.865（p<0.01）.Two methods detect that these protein expressions have a certain positive correlation with the malignant degree of lung cancer.However,the Western Blot method has the limitations of long detection process,cumbersome experiments,large reagent requirements,and low sample detection.Electrochemical immunosensors have the characteristics of high sensitivity,fast response time,low cost,simple equipment operation,and large-scale detection at one time.They have the value of development and application to clinical examination of tumor markers.