Prokaryotic Expression And Evaluation Of AHSG In Diagnosis Of Primary Hepatocellular Carcinoma

Alpha2-Heremans-Schmid glycoprotein (AHSG) is a kind of glycoprotein existing in human serum. The recent research indicated that the AHSG serum level in HCC patients was significantly higher than that in normal individuals, which suggested that it may be a serum biomarker for the diagnosis of primary hepatocelluar carcinoma. This study acquires the recombinat protein of AHSG in a prokaryotic expression way, which then was used as an antigen to detect the content of anti-AHSG antibody in different groups of people including HCC patients, patients with chronic liver diseases and normal individuals. In the subsequent study, the diagnostic value of AHSG will be evaluated alone or parallel combination with other TAAs as an indicator for primary hepatocelluar carcinoma.Methods1. Amplification of AHSG gene by RT-PCR:The total RNA was extracted from HepG2 cells. cDNA was synthesed with random primer by using total RNA as the template. Then, with the cDNA as a template, AHSG was amplified by polymerase chain reaction (PCR) using a pair of special primers designed according to the cDNA sequence, and the pair of primers included two restricted enzyme sites.2. Construction and characterization of recombinant expression vector:Firstly, the clone vector was constructed by ligating AHSG with pMD-18T vector and then transformed to DH5a. The clone vector was characterized by the way of restriction enzyme digestion and PCR amplification. The gene on the T vector was sequenced. Secondly, the AHSG gene in the clone vectors was cut off and then was ligated with pET-30a(+) plasmid to construct an expression plasmid, which was transformed into E. coli DH5a and validated by restriction enzyme digestion and PCR amplification.3. The recombinant expression plasmid was transformed into E. coli BL21.4. To find out the best IPTG concentration and induced time for the highest level expression of AHSG.5. The soluble analysis of protein:The expression form which include soluble protein and insoluble inclusion bodies of target protein in the massively induced products was analyzed, and to find the condition to improve the amount of soluble proteins.6. The purification of recombinant protein:The target protein with His tag was purified with Ni2+affinity chromatography. The concentration and the validity of the protein were detected by different methods.7. The serum level of anti-AHSG antibody was compared in different groups which included primary hepatocelluar carcinoma patients, chronic liver diseases patients and normal individuals after measured by enzyme linked immunosorbent assay (ELISA).8. The positive rates of anti-AHSG antibody in sera from patients with HCC group, chronic hepatitis and normal individuals were computed respectively on the basis of the pencentile P95 of normal group was as a cutoff point, then the diagnostic value of AHSG for HCC was evaluated with the method of screening trial.9. The best parallel combination was screened out and evaluated for the diagnosis of HCC from 14 kinds of TAAs which included AHSG, Calnuc, CDK2, cIAP, RalA, p62, p53, Cyclin B1, Koc, Imp 1, survivin, p16 and C-myc.ResultsThe prokaryotic expression system BL21(pET30a-AHSG) was constructed successfully; The most appropriate condition for the expression of target protein is 0.08mmol/L IPTG for 3h; The soluble analysis of protein indicated that the target protein mainly existed in the form of insoluble inclusion bodies; The inclusion bodies were dissolved best with 5mol/L urea among all the urea concentrations; Purified recombinant protein was acquired by Ni2+affinity chromatography and validated by Western blot.ELISA results showed that the anti-AHSG antibody level in primary hepatocelluar carcinoma group was higher than chronic liver diseases group or normal individuals. The sensitivity of AHSG to detect HCC was 13.1%, a low diagnostic value. The parallel combination of 9 TAAs can improve the sensitivity to 88.9%, the specificity to 86.8%. The positive predictive value, positive likelihood-ratio, negative predictive value, negative likelihood-ratio was 86.3%,6.80, 90.2%,0.12 respectively. Kappa value was 0.76, which indicated that the detected result of the screening trial was in accordance with true value moderately.ConclusionsThe prokaryotic expression system of AHSG and protein purification system were successfully constructed; The purified AHSG recombinant protein was acquired; The concentration of anti-AHSG antibody is improved in HCC patients and it can be as a serum biomarker for HCC, the diagnostic value for primary hepatocelluar carcinoma is low when used on its own while it is very high by a parallel combination of 9 TAAs including CyclinE, cIAP, CyclinBl, p53, P62, Calnuc, Koc and Impl.