Design of a chimaeric antigen and its use in the detection of IgG antibodies against rubella virus

Escherichia coli (E. coli) DH5α competent cells were obtained from our own laboratory. BL21 (DE3) competent cells was purchased from TransGen Biotech(China). T4 DNA ligase and all restriction enzymes were purchased from NEB. HRP-labelled goat anti-human IgG was purchased from Santa Cruz (USA). DAB (USA, Vector labs). Samples from 94 umbilical cord serum samples collected in 2014 from HuNan Province by the Chinese Center for Disease Control and Prevention. The origin of samplesis from individuals with history of 2B genotype infection. All of them were confirmed by a RV ELISA Kit (Germany, VirionSerion), of which 58 were positive and 36 were negative.

Based on references [12‐14], sequences with antigenic sites of E1 (199-286) and E2 (1-115) were selected, they were linked by a linker (GGGGSGGGGSGGGGS). After codon optimization, restriction endonuclease sites for BamHI and HindIII were added at both ends. The construct was then synthetized by the Invitrogen company (named P6T). P6T (18 ng/µL) was digested by BamHI and HindIII. P6T was purified and ligated to the pGEM-T expression vector. Then, the plasmid pGEM-T-P6T was transformed into E. coli DH5α. The positive clones were further identified with the restriction endonuclease digestion of BamHI and HindIII. The sequence was confirmed by SinoGenoMax Company.

Plasmid P6T was transformed to BL21 (DE3) competent cells. The transformed BL21 (DE3) was inoculated in LB medium containing 50 µg/mL ampicillin and induced expression. After cleaning twice with 10 mL high salt lysate (20 mM Tris, 0.5 m NaCl, 2 mM EDTA, 5% glycerol, 0.5% TritonX-100, PH8.0), ice water bath ultrasound (270 W, ultrasound 5S, pause 25S) for 15 min. After centrifugation, the precipitate was dissolved in 10 mL equilibrium buffer solution (20 mM Tris, 0.5 m NaCl, 10 mM imidazole, PH8.0), and centrifuged again. The resulting supernatant was filtered by a 0.45 um filter. The filtrated supernatant was purified by nickel column and eluted with gradient elution of 60 mmol/L and 300 mmol/L imidazole. The elution samples were analyzed by SDS-PAGE and the content and distribution of the target protein were observed [15]. The eluent with higher purity was desalted by dialysis, the target protein was concentrated by ultrafiltration centrifuge, and stored at 4 ℃ for later use.

Protein P6 was separated using 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% milk for 1 h at room temperature and were then washed 3 times in PBST. The membranes were then incubated with the cord blood serum at 1:200 dilution with the blocking solution at 4 °C overnight and washed 3 times. The membranes were then incubated with goat anti-human IgG-HRP-linked antibody (America, Santa Cruz Inc, 1:5 000) for 1 h at room temperature and washed 3 times.The protein levels were measured using a DAB Horseradish Peroxidase Color Development Kit.

After diluting the P6 sample with buffer solution, 100 µl of sample was added into each well, and the sample was coated at 4 °C for 12 h. Then, the plate was washed four times with PBST buffer. Then, 200 µl of blocking solution was added to each well, and the plate was stored at 37 °C for 2 h. The plate was washed with PBST buffer four times. Then, 100 µl of serum diluted with a blocking solution (PBST solution containing 5% BSA) was added. After being stored at 37 °C for 1 h, the plate was washed four times with PBST. Then, 100 µL of goat anti-human antibody (America, Santa Cruz Inc) diluted with blocking solution was added and incubated at 37 °C for 30 min. The plate was washed as described above. Then, 100 µL of TMB (America, KPL) was added to each well. The plate was stored in the dark for 5–15 min at RT. The optical density (OD) value at 450 nm (A450) was measured by microplate reader, and the OD value at 630 nm (A630) was used as a reference. The optimal conditions of serum dilution, antigen concentration and reaction time were established by performing a checkerboard titration. When the ratio of positive serum to negative serum A450s (P/N) was the best critical value, the coated antigen concentration was the best envelope concentration [16, 17].

Receiver operating characteristic (ROC) curve was drawn according to the OD value measured by the enzyme labelling method. The optimum critical value was determined by the Yoden index (Yoden index = sensitivity + specificity − 1). The sensitivity (Sensitivity = [true positive cases/(true positive cases + false negative cases)] × 100%) and specificity(Specificity = [true negative cases/(true negative cases + false positive cases)] × 100%) of the new detection method were calculated according to the critical value. SPSS18 software was used for statistical analysis of the experimental data.

The recombinant plasmid with multiple epitopes (named P6T) was constructed by selecting the immunoactive sites on the two structural proteins of RV. The P6T plasmid was approximately 666 bp (Fig. 1). The plasmid was successfully constructed and identified by sequencing and double restriction enzyme digestion (Fig. 1). P6T was expressed in E. coli, and the product was named P6. SDS-PAGE showed that the molecular weight of the protein P6 was approximately 44 kDa (Fig. 2). The expected fusion protein expressed encoded by just the pET-32a (+) vector alone would be around 20.4 kDa. In addition, the expected fusion protein expressed encoded by the P6T plasmid would be around 23.29 kDa (GSGLQPRADMAAPPAPPQPPRAHGQHYGHHHHQLPFLGHDGHHGGTLRVGQHHRNASDVLPGHWLQGGWGCYNLSDWHQGTHVCHTKHMDFWCVEHDRPPPATPTPLTTAANATTAAGGGGSGGGGSGGGGSDPGDLVEYIMNYTGNQQSRWGLGSPNCHGPDWASPVCQRHSPDCSRLVGATPERPRLRLVDADDPLLRTAPGPGEVWVTPVIGSQARK). The results showed that the protein P6 could be expressed in E. coli. The protein was expressed as inclusion body at 0.283 mg/mL.

Imidazole elution (60 mM, 150 mM, 300 mM) was used for the purification of the target protein. The results of SDS-PAGE showed that P6 protein was the purest with 150 mM imidazole concentration. It is more than 80% pure (Fig. 2).

The antigenicity of the protein P6 was identified by Western blotting. The results showed that the cord blood serum (RV-positive) as a first antibody and goat anti-human IgG-HRP as a second antibody showed a significant band at approximately 44 kDa. Moreover, RV-negative serum as a primary antibody showed no band (Fig. 2). No protein P6 was added, and no significant bands were observed with either RV-positive or RV-negative serum (Fig. 2). The results showed that the protein P6 could react specifically with RV-positive serum.

The optimal conditions of serum dilution, antigen concentration and reaction time were established. The purified P6 protein was used as antigen at an optimal concentration of 5 µg/ML. The use of purified antigen allowed the testing of sera at a 1:50 dilution without nonspecific reaction. Ninety-four samples of umbilical cord blood (VirionSerion ELISA method) were selected to verify the accuracy of this method (Table 1). An ROC chart was drawn according to the OD value, and the Yoden index was used to determine the best cut off (cut off = 0.317). According to the critical value (Fig. 3), the coincidence rate, specificity and sensitivity of the ELISA were 86.2%, 88.89%, 84.48% respectively.