A method for constructing and purifying a human cytomegalovirus multi-dominant epitope recombinant chimeric antigen

Abstract

The invention relates to the technical field of biological genetic engineering recombination, and in particular to a method for constructing and purifying a human cytomegalovirus multi-dominant epitope recombinant chimeric antigen. The method designs five strong dominant antigen epitopes, including aa595-614 and aa951-1048 of pp150 (UL32), aa202-433 of gp52 (UL44), aa297-510 of pp65 (UL83), and aa117-373 of pp38 (UL80a). The five strong dominant antigen epitopes are connected in series by flexible Linker connection, and a prokaryotic expression system is used to express a HCMV recombinant chimeric antigen with high specificity and affinity. The antigen can be applied to the development of in vitro diagnostic reagents, reduce false positives, false negatives and cross reactions in clinical detection, improve the detection rate and accuracy, and reach the level of imported detection reagents.

Description

Technical Field

[0001] The invention relates to the technical field of biological gene engineering recombination, and in particular to a method for constructing and purifying a human cytomegalovirus multi-dominant epitope recombinant chimeric antigen. Background Art

[0002] Human cytomegalovirus (HCMV) is a double-helix DNA virus of the β genus of the Herpesviridae family. Infection before and after pregnancy often leads to serious adverse consequences, such as miscarriage and developmental malformations, which seriously endangers human eugenics. With the development of society and the progress of science and technology, it is particularly important to establish a rapid, sensitive and specific detection method for HCMV and take early treatment measures for eugenics, eugenics and controlling the spread of HCMV.

[0003] The clinical detection method of HCMV is mainly based on immunological detection, which detects HCMV-IgG / IgM antibodies in human serum through immunological methods, and is used for auxiliary diagnosis of HCMV infection, evaluation of immune status and formulation of medical measures. At present, there are many types of HCMV antibody detection kits approved for registration by my country's Food and Drug Administration, but there is still a big gap in sensitivity and specificity between the detection results of domestic and imported kits. There are two main reasons: one is the reaction system, and the other is the antigen used. The antigen used is the basis for the development of in vitro diagnostic kits. Only when the specificity and affinity of the antigen are high, it is meaningful to optimize the detection system. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for constructing and purifying a recombinant chimeric antigen of human cytomegalovirus with multiple dominant epitopes, which can obtain a HCMV recombinant chimeric antigen with high specificity and affinity. The antigen can be applied to the development and production of domestic human cytomegalovirus diagnostic reagents, reduce false positives, false negatives and cross reactions in clinical detection, and improve the detection rate and accuracy.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A method for constructing and purifying a human cytomegalovirus multi-dominant epitope recombinant chimeric antigen comprises the following steps:

[0007] S1. Design five strong dominant antigenic epitopes, including aa595-614 and aa951-1048 of HCMV virus pp150 protein (UL32), aa202-433 of HCMV virus gp52 protein (UL44), aa297-510 of HCMV virus pp65 protein (UL83), and aa117-373 of HCMV virus pp38 protein (UL80a), and connect the five strong dominant antigenic epitopes in series at the Kpn I / Xhol I restriction site of pET32a expression plasmid through a flexible linker (ggc ggc ggc ggc agc), and then perform Escherichia coli preferred codon analysis on the target gene fragment sequence through biological software, convert the nucleic acid sequence into Escherichia coli preferred synonymous codons, and obtain an optimized gene sequence, and the optimized gene sequence is shown in SEQ ID NO: 1;

[0008] S2, inserting the optimized gene sequence into the Kpn I / Xho I restriction site of the pET32a plasmid for gene recombination to construct the HCMV / pET32a plasmid;

[0009] S3, transforming the HCMV / pET32a plasmid into the BL21(DE3) expression bacteria to construct the expression bacteria CMV / pET32a / BL21(DE3);

[0010] S4, inducing the expression of the expression bacteria CMV / pET32a / BL21(DE3) with IPTG, and collecting the bacteria;

[0011] S5, lysing and denaturing the collected whole bacterial cells;

[0012] S6, nickel column affinity purification;

[0013] S7. Dialysis and renaturation to harvest HCMV recombinant antigen.

[0014] Furthermore, in the step S5, the collected whole bacterial cells are added with a lysis solution at a mass volume ratio of 1:15 to 1:30, the lysis solution components are 6M guanidine hydrochloride or 8M urea + 20mM PB + 1-3mM imidazole + 1-3mM DTT, pH = 8.0, and ultrasonically disrupted in an ice bath, with an output power of 90%, working for 3s, an interval of 5s, and ultrasonication for 20-30min. The supernatant is collected by centrifugation at 10000-12000rpm for 30min and sterilized by 0.22μm filter membrane.

[0015] Furthermore, the step S6 includes:

[0016] (1) NI-NTA nickel column flows through 10 to 20 column volumes of purified water;

[0017] (2) The lysate was equilibrated for 10 to 20 column volumes until the baseline was flat;

[0018] (3) Low temperature cycle loading for 1 to 3 hours;

[0019] (4) Use 8 M urea + 20 mM PB + 5-10 mM imidazole + 1 mM 2-Me as washing solution for 10-20 column volumes;

[0020] (5) Use 8 M urea + 20 mM PB + 250-400 mM imidazole + 1 mM 2-Me as the eluent for 10 column volumes and collect the elution peak, which is the denatured HCMV purified antigen.

[0021] Furthermore, in the step S7, the elution peak sample is loaded into a dialysis bag with a cutoff of 10 to 30 kD, placed in 20 to 50 times the volume of the refolding solution, and dialyzed at 4°C overnight. The refolding solution without GSH / GSSG is used for a second dialyzation every day, and the solution is changed every 3 to 5 hours under magnetic stirring. After the third change of the solution, the solution is allowed to stand at 4°C overnight for sufficient dialysis.

[0022] Furthermore, the refolding solution is: 20mM Tris+1mM EDTA+50-100mM urea+0.2mM GSH+0.1mM GSSG+2%-5% glycerol, pH=8.5.

[0023] Furthermore, in step S7, the liquid in the dialysis bag is collected, centrifuged at 10,000-12,000 rpm for 20 min, the precipitate is discarded, the supernatant is collected, and the supernatant is sterilized by filtering at 0.22 μm. After packaging, the supernatant is frozen and stored at -80°C or freeze-dried.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention connects the five-segment advantage table of human cytomegalovirus (HCMV) in series using a flexible linker, and uses a prokaryotic expression system to express a HCMV recombinant chimeric antigen with high specificity and affinity. The antigen is completely independently designed, has low production and purification costs, strong specificity, and high sensitivity. It can be applied to the development and production of domestic human cytomegalovirus diagnostic reagents, reduce false positives, false negatives and cross reactions in clinical detection, and improve the detection rate and accuracy.

[0026] The chimeric antigen of the five dominant epitopes of HCMV prepared by the present invention has a high induced expression amount, is easy to purify and renature, and has a high renaturation rate. The antigenicity evaluation of clinical samples is tested, and the specificity reaches 100%, and the sensitivity and accuracy can reach more than 99%, which can reach the level of imported detection reagents and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1Schematic diagram of the insertion position of the gene sequence.

[0028] Figure 2 The results are for sequencing of 1-130bp sequences.

[0029] Figure 3 The sequencing results are 2370-2541bp.

[0030] Figure 4 is the result of enzyme digestion identification;

[0031] In the figure: 1- double enzyme digested plasmid; 2-Marker; 3- undigested control plasmid.

[0032] Figure 5 The electrophoresis results of HCMV expression at different temperatures;

[0033] In the figure: 1-37℃ whole bacteria; 2-30℃ whole bacteria; 3-20℃ whole bacteria; 4-20℃ ultrasonic supernatant; 5-30℃ ultrasonic supernatant; 6-37℃ ultrasonic supernatant; 7-Maker; 8-20℃ ultrasonic precipitation; 9-30℃ ultrasonic precipitation; 10-37℃ ultrasonic precipitation.

[0034] Figure 6 The electrophoresis results for purified HCMV;

[0035] In the figure: 1-Purification of HCMV; 2-Maker. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] In order to obtain a highly specific HCMV recombinant chimeric antigen that can be used for the development of in vitro diagnostic reagents for detecting HCMV antibodies, the inventors first designed five strong dominant antigenic epitopes, including aa595-614 and aa951-1048 of pp150 (UL32), aa202-433 of gp52 (UL44), aa297-510 of pp65 (UL83), and aa117-373 of pp38 (UL80a), by comparing literature and the dominant antigenic epitopes provided by major in vitro diagnostic reagent (IVD) raw material manufacturers. The five strong dominant antigenic epitopes were connected in series to the Kpn I / Xhol I restriction site of the pET32a expression plasmid through a flexible linker (ggc ggc ggc ggc agc) to construct an HCMV / pET32a plasmid. The sequence insertion position is as follows: Figure 1 As shown, the target gene fragment sequence is then analyzed for E. coli preferred codons using biological software, the nucleic acid sequence is converted into E. coli preferred synonymous codons, and the optimized gene sequence is obtained:

[0038]

[0039] Next, the HCMV / pET32a plasmid was transformed into the BL21(DE3) expression bacteria to construct the expression bacteria CMV / pET32a / BL21(DE3); the plasmids were extracted from the expression bacteria CMV / pET32a / BL21(DE3) and the DH5α clone bacteria and sequenced. The results showed that the base sequence was consistent with the designed sequence. Since the sequencing results were too long, the sequencing results of 1-130bp and 2370-2541bp were intercepted for display, as shown in FIG. Figure 2 and Figure 3 After plasmid extraction, Kpn I / Xho I double digestion was performed, and agarose gel electrophoresis was used for identification. A brighter target band (the full length of the target band is 2541 bp) was observed below 3000 bp, and the control plasmid without enzyme digestion was established, proving that the target fragment was correctly inserted into the plasmid. The results are shown in Figure 4 shown.

[0040] Then, the expression bacteria CMV / pET32a / BL21(DE3) were induced by IPTG and the cells were collected. By comparing the induction expression at 37℃, 30℃ and 20℃, it was found that the expression amount increased significantly when the temperature was lowered, such as Figure 5 shown.

[0041] Then, the collected whole bacteria were lysed and denatured, and then subjected to nickel column affinity purification and dialyzed for renaturation to harvest the HCMV recombinant antigen.

[0042] Embodiment 1:

[0043] 1. Activation: After the glycerol bacteria HCMV / pET32a / BL21 (DE3) strain that has been successfully identified and expressed in the preliminary experiment is melted in an ice bath, 10 μL of the strain is taken out and added to 10 mL LB medium (containing 100 μg / mL ampicillin), and activated at 37°C, 200 rpm overnight.

[0044] 2. Fermentation and induction: 10 mL of activated bacterial solution was added to 1 L of liquid LB medium for expansion culture. The culture conditions were 200 rpm and 37 °C for 4 h. After the culture was completed, a final concentration of 0.1 mM IPTG was added to the bacterial solution for induction expression. The conditions were 200 rpm and 20 °C for induction overnight.

[0045] 3. Bacteria collection: After induction, centrifuge at 6000 rpm for 30 min to collect the bacteria, resuspend in TE buffer (20 mM Tris + 1 mM EDTA, pH = 8.5), wash once by centrifugation at 6000 rpm for 30 min, and then weigh the wet weight of the bacteria.

[0046] 4. Whole-bacterial lysis: The harvested whole-bacterial cells were added with lysis buffer at a mass-to-volume ratio of 1:20. The lysis buffer consisted of 6 M guanidine hydrochloride + 20 mM PB + 2 mM imidazole / 3 mM DTT (pH = 8.0). Ultrasonic disruption was performed under ice bath with an output power of 90%, working for 3 s and an interval of 5 s. After ultrasonication for 20 min, the supernatant was collected by centrifugation at 10,000 rpm for 30 min and sterilized by filtration with a 0.22 μm filter membrane.

[0047] 5. Nickel column purification:

[0048] (1) 10 column volumes of purified water were used for the Ni-NTA nickel column;

[0049] (2) The lysate was equilibrated for 10 column volumes until the baseline was flat;

[0050] (3) Low temperature cycle loading for 2 h;

[0051] (4) Washing solution (8 M urea + 20 mM PB + 7 mM imidazole + 1 mM 2-Me pH = 8.0) for 10 column volumes;

[0052] (5) Elution with elution buffer (8 M urea + 20 mM PB + 250 mM imidazole + 1 mM 2-Me pH = 8.0) for 10 column volumes, and the elution peak is collected as the denatured HCMV purified antigen, and electrophoresis is performed. The results are as follows: Figure 6 Shown: Purity> 90%.

[0053] 6. Dialysis renaturation: The eluted peak sample was loaded into a dialysis bag with a cutoff of 10 kD, placed in 20 times the volume of renaturation solution (20 mM Tris + 1 mM EDTA + 79 mM urea + 0.2 mM GSH + 0.1 mM GSSG + 5% glycerol pH = 8.5), and dialyzed overnight at 4 ° C. This step is a slow renaturation and can increase the renaturation rate. Magnetic stirring should not be used. The next day, a second dialysis was performed using a renaturation solution without GSH / GSSG. The solution was changed every 4 hours with magnetic stirring. After the third change of solution, the solution was allowed to stand at 4 ° C overnight for sufficient dialysis.

[0054] 7. Harvest: Collect the liquid in the dialysis bag, centrifuge at 10000rpm for 20min, discard the precipitate and collect the supernatant, filter and sterilize at 0.22μm, and store frozen at -80℃ or freeze-dried after packaging. The protein concentration of the purified antigen was detected to be 1.33mg / mL, and the purity was 91.06%.

[0055] Embodiment 2:

[0056] 1. Activation: After the glycerol culture was melted in an ice bath, 5 μL of the culture was added to 10 mL of LB medium (containing 50 μg / mL ampicillin) and activated at 37°C, 200 rpm overnight.

[0057] 2. Fermentation and induction: 10 mL of activated bacterial solution was added to 0.8 L of liquid LB medium for expansion culture. The culture conditions were 200 rpm and 37 °C for 5 h. After the culture was completed, a final concentration of 0.2 mM IPTG was added to the bacterial solution for induction expression. The conditions were 200 rpm and 16 °C for induction overnight.

[0058] 3. Bacteria collection: After induction, centrifuge at 8000 rpm for 30 min to collect the bacteria, resuspend in PBS buffer (20 mM, pH = 8.0), wash once by centrifugation at 8000 rpm for 30 min, and then weigh the wet weight of the bacteria.

[0059] 4. Whole-bacterial lysis: The harvested whole-bacterial cells were added with lysis buffer at a mass-to-volume ratio of 1:30. The lysis buffer consisted of 8 M urea + 20 mM PB + 2 mM imidazole + 2 mM DTT (pH = 8.0). Ultrasonic disruption was performed under ice bath with an output power of 90%, working for 3 s, with an interval of 5 s. After ultrasonication for 30 min, the supernatant was collected by centrifugation at 12000 rpm for 30 min and sterilized by filtration with a 0.22 μm filter membrane.

[0060] 5. Nickel column purification:

[0061] (1) NI-NTA nickel column with purified water for 20 column volumes;

[0062] (2) The lysate was equilibrated for 20 column volumes until the baseline was flat;

[0063] (3) Low temperature cycle loading for 3 h;

[0064] (4) Washing solution (8 M urea + 20 mM PB + 10 mM imidazole + 1 mM 2-Me pH = 8.0) for 20 column volumes;

[0065] (5) Elution with elution buffer (8 M urea + 20 mM PB + 400 mM imidazole + 1 mM 2-Me pH = 8.0) for 10 column volumes, and the elution peak is collected as the denatured HCMV purified antigen, and electrophoresis is performed. The results are as follows: Figure 6 Shown: Purity> 90%.

[0066] 6. Dialysis renaturation: The eluted peak sample was loaded into a dialysis bag with a cutoff of 30 kD, placed in 50 times the volume of renaturation solution (20 mM Tris / 1 mM EDTA + 100 mM urea + 0.2 mM GSH + 0.1 mM GSSG + 2% glycerol pH = 8.5), and dialyzed overnight at 4 ° C. This step is a slow renaturation and can increase the renaturation rate. Magnetic stirring should not be used. The next day, a second dialysis was performed using a renaturation solution without GSH / GSSG. The solution was changed every 5 hours with magnetic stirring. After the third change of solution, the solution was allowed to stand overnight at 4 ° C for sufficient dialysis.

[0067] 7. Harvest: Collect the liquid in the dialysis bag, centrifuge at 12000rpm for 20min, discard the precipitate and collect the supernatant, filter and sterilize at 0.22μm, and store frozen at -80℃ or freeze-dried after packaging. The protein concentration of the purified antigen was detected to be 1.15mg / mL and the purity was 92.27%.

[0068] Embodiment 3:

[0069] 1. Activation: After the glycerol culture was melted in an ice bath, 20 μL of the culture was added to 10 mL of LB medium (containing 100 μg / mL ampicillin) and activated at 37°C, 200 rpm overnight.

[0070] 2. Fermentation and induction: 10 mL of activated bacterial solution was added to 1.5 L of liquid LB medium for expansion culture. The culture conditions were 200 rpm and 37 °C for 3 h. After the culture was completed, a final concentration of 0.3 mM IPTG was added to the bacterial solution for induction expression. The conditions were 200 rpm and 18 °C for induction overnight.

[0071] 3. Bacteria collection: After induction, centrifuge at 7000 rpm for 30 min to collect the bacteria, resuspend in TE buffer (20 mM Tris + 1 mM EDTA, pH = 8.5), wash once with centrifugation at 7000 rpm for 30 min, and then weigh the wet weight of the bacteria.

[0072] 4. Whole-bacterial lysis: The harvested whole-bacterial cells were added with lysis buffer at a mass-to-volume ratio of 1:25. The lysis buffer consisted of 6 M guanidine hydrochloride + 20 mM PB + 1 mM imidazole + 1 mM DTT (pH = 8.0). Ultrasonic disruption was performed under ice bath with an output power of 90%, working for 3 s, with an interval of 5 s. After ultrasonication for 25 min, the supernatant was collected by centrifugation at 11000 rpm for 30 min and sterilized by filtration with a 0.22 μm filter membrane.

[0073] 5. Nickel column purification:

[0074] (1) NI-NTA nickel column with 15 column volumes of purified water;

[0075] (2) The lysate was equilibrated for 15 column volumes until the baseline was flat;

[0076] (3) Low temperature cycle loading for 1 h;

[0077] (4) Washing solution (8 M urea + 20 mM PB + 5 mM imidazole + 1 mM 2-Me pH = 8.0) for 15 column volumes;

[0078] (5) Elution with elution buffer (8 M urea + 20 mM PB + 300 mM imidazole + 1 mM 2-Me pH = 8.0) for 10 column volumes, and the elution peak is collected as the denatured HCMV purified antigen, and electrophoresis is performed. The results are as follows: Figure 6Shown: Purity> 90%.

[0079] 6. Dialysis renaturation: The eluted peak sample was loaded into a dialysis bag with a cutoff of 20 kD, placed in 30 times the volume of renaturation solution (20 mM Tris + 1 mM EDTA + 80 mM urea + 0.2 mM GSH + 0.1 mM GSSG + 3% glycerol pH = 8.5), and dialyzed overnight at 4 ° C. This step is a slow renaturation and can increase the renaturation rate. Magnetic stirring should not be used. The next day, a second dialysis was performed using renaturation solution without GSH / GSSG. The solution was changed every 3 hours with magnetic stirring. After the third change of solution, the solution was allowed to stand at 4 ° C overnight for sufficient dialysis.

[0080] 7. Harvest: Collect the liquid in the dialysis bag, centrifuge at 11000rpm for 20min, discard the precipitate and collect the supernatant, filter and sterilize with 0.22μm, and store frozen at -80℃ or freeze-dried after packaging. The protein concentration of the purified antigen was detected to be 0.96mg / mL and the purity was 90.83%.

[0081] Test data

[0082] The purified HCMV antigen was used to prepare Elisa reagents, which were compared with imported similar reagents to detect HCMV-IgG antibodies in clinical serum samples. The detection system and operation steps were as follows:

[0083] 1. Coating: Dilute the purified target protein to 1 μg / mL with sodium carbonate-sodium bicarbonate buffer, add 100 μL to each well of the ELISA plate, and place at 4°C for coating overnight;

[0084] 2. Blocking: The next day, pour out the coating solution in the ELISA plate, wash the plate 3 times with PBST (Note: try to pat dry the residual washing solution after each wash); block with 2% BSA-PBST blocking solution, 200 μL / well, 37℃2h (or 4℃overnight);

[0085] 3. Primary antibody incubation: After blocking, discard the blocking solution and wash the plate three times with PBST; dilute the anti-HCMV IgG negative and positive serum samples to be tested 1:100 with 1% BSA in PBST, add 100 μL / well of the diluted serum (primary antibody) to the washed ELISA plate, and incubate at 37°C for 1 hour;

[0086] 4. Secondary antibody incubation: After the primary antibody incubation is completed, the primary antibody is discarded and the plate is washed 3 times with PBST; HRP-labeled mouse anti-human IgG monoclonal antibody (secondary antibody) is diluted with 1% BSA-PBST at a ratio of 1:10000, added to the ELISA plate, 100 μL / well, and incubated at 37°C for 1 hour;

[0087] 5. TMB color development: After the secondary antibody incubation is completed, pour out the secondary antibody and wash the plate 3 times with PBST; add TMB color development solution to the wells of the ELISA plate in the dark, 100 μL / well, after color development for 15 minutes, add 50 μL / well of stop solution (2 mol / L H2SO4) to stop color development;

[0088] 6. Detection: Measure the OD450 / 630 readings with an ELISA reader within 20 minutes and save the results.

[0089] The results are shown in Table 1.

[0090] Table 1. Comparison of homemade Elisa reagents and imported reagents

[0091]

[0092]

[0093] Calculated from Table 1: Sensitivity = a / (a+c)×100%=99.1%; Specificity = d / (b+d)×100%=100%; Accuracy = (a+d) / n×100%=99.1%. It can be seen that the HCMV antigen detection results of the present invention and the imported test kit detection results in a large number of sample detections can reach a consistency rate of more than 99%, and the enzyme-linked immunosorbent assay method established by the solid phase carrier coating thereof has a sensitivity and specificity close to the level of imported detection reagents. If the detection system of the homemade test kit is further optimized, the interference factors in the sample are reduced, and the sensitivity of the homemade test kit is improved, the consistency rate can be further improved.

[0094] Only the preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in this field without departing from the purpose of the present invention, and various changes should be included in the protection scope of the present invention.

Claims

1. A method for constructing and purifying a multi-dominant epitope recombinant chimeric antigen of human cytomegalovirus (HCMV), characterized in that: The steps include: S1. Design five strong dominant antigen epitopes, including aa595-614 and aa951-1049 of pp150 (UL32), aa202-433 of gp52 (UL44), aa297-510 of pp65 (UL83), and aa117-373 of pp38 (UL80a), and connect the five strong dominant antigen epitopes in series at the Kpn I / Xhol I restriction site of the pET32a expression plasmid through a flexible linker, and then convert the nucleic acid sequence into the preferred synonymous codons of Escherichia coli, and obtain the optimized gene sequence as shown in SEQ ID NO: 1; S2, inserting the optimized gene sequence into the Kpn I / Xho I restriction site of the pET32a plasmid for gene recombination to construct the HCMV / pET32a plasmid; S3, transform the HCMV / pET32a plasmid into the BL21 (DE3) expression bacteria to construct the expression bacteria CMV / pET32a / BL21 (DE3); S4, inducing the expression of the expression bacteria CMV / pET32a / BL21 (DE3) with IPTG, and collecting the bacteria; S5, lysing and denaturing the collected whole bacterial cells; S6, nickel column affinity purification; S7. Dialysis and renaturation to harvest HCMV recombinant antigen.

2. The method for constructing and purifying a multi-dominant epitope recombinant chimeric antigen of human cytomegalovirus (HCMV) according to claim 1, characterized in that: In the step S5, the collected whole bacterial cells are added to a lysis solution at a mass volume ratio of 1:15 to 1:30, the lysis solution components are 6 M guanidine hydrochloride or 8 M urea + 20 mM PB + 1-3 mM imidazole + 1-3 mM DTT, pH = 8.0, and ultrasonically disrupted in an ice bath, with an output power of 90%, working for 3 s, and an interval of 5 s. After ultrasonication for 20 to 30 min, the supernatant is collected by centrifugation at 10000-12000 rpm for 30 min, and sterilized by filtration with a 0.22 μm filter membrane.

3. The method for constructing and purifying a human cytomegalovirus (HCMV) multi-dominant epitope recombinant chimeric antigen according to claim 1, characterized in that: The step S6 includes: (1) NI-NTA nickel column flows through 10 to 20 column volumes of purified water; (2) Equilibrate the lysate for 10 to 20 column volumes until the baseline is flat; (3) Low temperature cycle loading for 1 to 3 h; (4) Use 8 M urea + 20 mM PB + 5-10 mM imidazole + 1 mM 2-Me as washing solution for 10-20 column volumes; (5) Use 8 M urea + 20 mM PB + 250-400 mM imidazole + 1 mM 2-Me as the eluent for 10 column volumes and collect the elution peak, which is the denatured HCMV purified antigen.

4. The method for constructing and purifying a human cytomegalovirus (HCMV) multi-dominant epitope recombinant chimeric antigen according to claim 1, characterized in that: In the step S7, the eluted peak sample is loaded into a dialysis bag with a cutoff of 10 to 30 kD, placed in a 20 to 50 times volume of refolding solution, and dialyzed at 4°C overnight. The refolding solution without GSH / GSSG is used for a second dialysis every day, and the solution is changed every 3 to 5 hours with magnetic stirring. After the third change of solution, the sample is allowed to stand at 4°C overnight for full dialysis.

5. The method for constructing and purifying a human cytomegalovirus (HCMV) multi-dominant epitope recombinant chimeric antigen according to claim 4, characterized in that: The refolding solution is: 20 mM Tris + 1 mM EDTA + 50-100 mM urea + 0.2 mM GSH + 0.1 mM GSSG + 2%-5% glycerol, pH=8.

5.

6. The method for constructing and purifying a human cytomegalovirus (HCMV) multi-dominant epitope recombinant chimeric antigen according to claim 4, characterized in that: In step S7, the liquid in the dialysis bag is collected, centrifuged at 10,000 to 12,000 rpm for 20 min, the precipitate is discarded, the supernatant is collected, and the supernatant is sterilized by 0.22 μm filtration. After aliquoting, the supernatant is stored frozen at -80°C or freeze-dried.

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