| Objective:To purify two soluble hepatocellular carcinoma-associated antigens SMP30 and CNN2 through high expression of genetically engineered bacteria, and to detect the relevant antibody expression in the serum of patients with liver diseases like hepatocellular carcinoma and hepatitis with hepatic cirrhosis as well as normal persons by taking these two genetically engineered proteins as antigens, thus exploring the value of primary hepatic cancer detection by integrating the two antibodies in the serum with AFP.Methods:The genetically engineered bacteria E.Coli.BL21 were activated and cultured on the medium plate with LB-Kan+at 37℃ for overnight. Single colon was selected and inoculated in LB-Kan+culture solution at 37℃ for overnight. The strain was preserved. According to the proportion of 1:100 for strain and medium, we inoculated the strain in a new large-volume medium solution with LB-Kan+ for 2.5 hours. After that, the IPTG inducer was added in for 5 hours of induction expression at 37℃. The cell was centrifuged with the method of ultrasound bacteria breaking, and the supernatant and sediment were selected for SDS-PAGE gel electrophoresis. The supernatant was loaded on the Ni-NTA affinity column and eluted with different gradients of imidazole solution. The eluents were concentrated by ultrafiltration using ultra-filter tube. The purified protein was analyzed and identified with the method of SDS-PAGE. We took the SMP30 and CNN2 to coat the ELISA plate. The indirect ELISA method was adopted to detect the existence of relevant antibodies in the serum of patients with primary HCC, patients with hepatitis cirrhosis, and normal people. Statistical analyses were carried out by integrating various clinical indexes. Through the combination of data, telephone follow-up was also conducted for positive subjects and partial negative subjects in the hepatitis cirrhosis group, so as to analyze whether those people got the disease of liver cancer.Results:The supernatant and sediment of genetically engineered bacteria before and after induction with the IPTG were analyzed by SDS-PAGE. The result showed that there were significant differences for aimed protein band before and after induction. Aimed protein band could be seen obviously thicker in the supernatant and sediment of the induced bacterial liquid than that of the uninduced bacterial liquid. According to analyses, a large number of target proteins were expressed by the genetic engineering bacteria E.Coli.BL21 after IPTG induction, and part of them belonged to the soluble protein. The result was rather consistent with the protein expressed and purified by the research group previously. The supernatant covering the column was extracted for purification, and its concentrated protein after ultrafiltration in the ultra-filter tube was obtained. Through the analysis of SDS-PAGE electrophoresis, the target protein whose band is relatively single was obtained. Analyzed by ImageJ software, the protein purity of SMP30 was 94.13%, and that of CNN2 was 81.7%. Both proteins were taken as antigen coated ELISA plates. As could be learnt from the indirect ELISA analysis, the positive rate for the serum antibody to SMP30 of HCC group and hepatitis cirrhosis group was 25.87% and 4.25% respectively, and no positive sample was detected for the normal person group. The positive rate for the serum antibody to CNN2 of HCC group and hepatitis cirrhosis group was 21.3% and 9.9% respectively, and the positive detection rate for the normal person group was 1.2%. The receiver operating characteristic (ROC) curve was adopted for analysis. When the HCC group was diagnosed by SMP30, the area under the curve of ROC was 0.824, the sensitivity was 25.87%, and the specificity was 97.45%. When the HCC group was diagnosed by CNN2, the area under the curve of ROC was 0.73, the sensitivity was 21.14%, and the specificity was 94.30%. With the method of pearson Chi-Square bilateral detection, Chi-Square test was conducted for indexes of the HCC group of both proteins. The result showed that the P value of each group was larger than 0.05, indicating there was no statistical significance. Therefore, it was still uncertain if the positive serum was relevant with the clinical data temporarily. The HCC serum was detected by both proteins combining with the AFP. The positive detection rate was 73.98%, which was dramatically higher than the result of single detection. Through the statistical analysis, P< 0.05, so the difference was with statistical significance. The method of joint detection could help to improve the diagnosis rate of primary HCC and to reduce missed diagnosis. Telephone follow-up for positive samples and partial negative samples in the hepatitis cirrhosis group was conducted, and no liver cancer was discovered temporarily. The relationship between the morbidity of liver cancer and the two relevant antigens could not be identified.Conclusions:1. Our research shows that the anti-SMP30/CNN2 antibody positive rate in the serum of HCC patients is significantly higher than hepatitis patients with hepatic cirrhosis and health individuals, which is of certain research values for HCC detection but still cannot be taken as an index for clinical diagnosis of HCC independently.2. SMP30 and CNN2 were combined with AFP for combined assay, and the positive rate of the antibody in the HCC serum detected by this means was higher than that by single detection. Combined assay could contribute to the enhancement of the diagnosis rate of primary HCC as well as the reduction of missed diagnosis.3. According to the telephone follow-up result, the relationship between the serum antibody positive of SMP30 and CNN2 and the morbidity of liver cancer could not be identified temporarily. |
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