Design of a chimeric antigen containing multiple immunodominant epitopes and its use in detection of IgM antibodies against Rubella virus / 生物工程学报

A chimeric antigen designated B103 containing six immunodominant regions derived from three structural proteins of Rubella virus (RV) was designed and its utility in serological diagnosis was assessed. Protein B103 is comprised of aa 1-30 & aa 96-123 of C protein, aa 31-105 of E2 protein, as well as aa 11-39, aa 154-277 & aa 389-412 of E1 protein. In addition, it contains thioredoxin (TRX) at the N-terminal and His tag at the C-terminal. B103 was expressed in Escherichia coli BL21(DE3) and purified by Streamline Chelating affinity and DEAE anion exchange chromatography. Based on the antigenicity of B103 as verified by Western blotting analysis, we constructed and evaluated a novel capture ELISA for RV-IgM detection. B103 was expressed in a soluble form, accounting for 18.57% of the total bacterial proteins. After purification, the concentration and purity of protein B103 were 3.026 mg/mL and 95.35%, respectively. Western blotting analysis demonstrated that protein B103 could react with acute-phase serum of RV. By ELISA, 40 negative sera and 40 RV-acute phase sera were detected. The sensitivity, specificity, positive predictive value, negative predictive value and coincidence rate of the ELISA were 92.50%, 95.00%, 94.87%, 92.68% and 93.75%, respectively. The McNemer analysis suggested that there was no statistical difference between the 'Gold standard' and the novel ELISA with a kappa coefficient of 0.900, indicating excellent consistency. B103 chimeric protein with excellent antigenicity obtained from prokaryotic expression followed by chromatography purification could prove useful for early diagnosis of RV infection.

Preparation of multi-epitope recombinant diagnostic antigen of Mycobacterium tuberculosis / 生物工程学报

Multi-epitope recombinant diagnostic antigen (designated 'B102') of Mycobacterium tuberculosis (Mtb) was prepared and evaluated as a serological diagnostic antigen. With TRX at the N-terminal and His tag at the C-terminal, the multi-epitope Mtb recombinant diagnostic antigen including 11 predicted B-cell epitopes from 6 Mtb antigens (PstS1, ESAT6, CFP10, Ag85B, Ag85A and PPE54) was expressed in Escherichia coli BL21 (DE3) and purified by Ni²⁺-Chelating affinity and DEAE anion exchange chromatography. Based on the antigenicity of B102 confirmed in Western blotting analysis, we constructed and evaluated a double-antigen sandwich ELISA for diagnosis of Mtb infection. The protein B102 exists in the form of inclusion bodies, accounting for 31.25% of the total proteins of the bacteria. After purification and renaturation, protein B102 exists in soluble form with the concentration 3.124 mg/mL and the homogeneity 96.71%. WB analysis demonstrated that protein B102 could react with antibodies in Mtb positive serum. Using the novel antigen in ELISA, we tested 60 Mtb-related positive and negative serum; The results showed the sensitivity, specificity, positive and negative predictive values and coincidence rate of the detection method is 90.00%, 93.33%, 93.10%, 90.32% and 91.67%, respectively. The McNemer analysis suggested there was no statistical difference between the 'Gold standard' and the novel ELISA with kappa 0.833, which suggested the excellent consistency. By prokaryotic expression and chromatography purification, the multi-epitope recombinant antigen B102 was obtained with excellent antigenicity, which could be applied for Mtb-related serological detection.

Preparation of peptide mimotope-based diagnostic antigen of Epstein-Barr virus infection / 中华实验和临床病毒学杂志

Objective@#To prepare peptide minotope-based recombinant diagnostic antigen of Epstein-Barr virus (EBV) infection and evaluate its antigenicity preliminarily.@*Methods@#With Trx at the N-terminal and His tag at the C-terminal, the peptide minotope of EBV (GP125, F1, A2, A3C2) was expressed in Escherichia coli and purified by affinity and anion exchange chromatography (designated 'H58’); based on antigenicity of H58 identified by Western blotting (WB), we constructed and evaluated a novel early diagnostic ELISA for EBV infection.@*Results@#The soluble H58 protein with high concentration (2.8 mg/ml) and purity (99.01) was obtained; WB analysis found that there was an obvious band (28 ×103) on the NC membrane, using H58 anti-Trx monoclonal antibody or acute-phase sera of EBV infection as the first antibody. With the novel ELISA, 50 positive sera of EBV infection and 50 negative sera were detected, displaying that the grouping of OD value of positive serum (95%CI: 1.233-1.489) and negative serum (95%CI: 0.113-0.159) was different (P<0.05) with the sensitivity 98.0%, specificity 96.0% and kappa value 0.940.@*Conclusions@#By E. coli expression and affinity and ion exchange chromatography purification, the peptide minotope-based recombinant diagnostic antigen of EBV infection was obtained with excellent antigenicity, which could be applied for serological detection of EBV infection.

Study on the purification and binding activity of the sodium channel / 中国药理学通报

Aim To apply sodium channels isolated and purified from rat brain and rat skelatal muscle sarcolemma to the study conotoxin.Methods Crude isolations of sodium channels were purified by 3 kinds of chromatography.The interaction between the sodium channels and conus betulinus was studied by the ligand binding experiment.Result The sequential chromatography resulted in 370-fold purification from rat brain and 2436-fold purification from rat skeletal muscle.Average specific binding active sites of purified sodium channels increased to 321 nmol?g-1 for rat brain and 268 nmol?g-1 for rat skeletal muscle respectively;Studying the interaction between venom from conus betulinus and the purified sodium channels indicated the fraction Ⅴ8 showed the highest affinity for the binding sites of the purified sodium channels.Conclusion Rat brain sodium channel and rat skeletal muscle sodium channel were purified to high purity;the fraction Ⅴ8 has the similar effect to that of the traditional ?-conotoxin.

Isolation, Purification and its antitumor activity of glycosaminoglycan from Pinctada martensii / 中国海洋药物

Crude glycosaminoglycan(CPG) from the whole viscera of Pinctada martensii was isolated with the two-enzyme hydrolysis and purified by the chromatograph.And the antitumor activity was studied with MTT colormetric assay. The CPG could be separated into two fractions(GAG-1, GAG-2) by the DEAE-52-cellulose ion exchange chromatograph. The contents of hexosamine of CPG,GAG-1, GAG-2 were 54.6%,50.0%,35.4%,respectively. The sulfate of GAG-2 was 13.8%. The inhibiting rates of CPG, GAG-1 and GAG-2 to HL-60 cells were 54.6%, 50.0% and 35.4%,respectively. The inhibition to HL-60 of 5-Fu could be increased by combined use with CPG, GAG-1 and GAG-2, GAG-1 was the main active fraction.