[0021] The present invention will be further described in detail below in conjunction with the drawings and embodiments.
[0022] (1) Construction of a pHMOXG-alpha-ZKE recombinant expression vector containing Zika virus structural protein E gene fragment 1. According to the reported Zika virus structural protein E gene information (GenBank: KU820899.2), artificially design the Zika to be expressed Virus structural protein E gene fragment (designed in May 2017), the two ends of which use amplification primers (primer 1 and primer 2, designed in July 2017) can add restriction site EcoRI (carbon end, see sequence underlined) Position) and restriction site NotI (nitrogen end, see sequence italic position). The sequence of the artificially designed Zika virus structural protein E gene fragment is shown in SEQ.ID.NO.1, which is artificially synthesized.
[0023] 2. After successfully transferring the plasmid (pMD18-T, Takara) containing the designed Zika virus structural protein E gene fragment into Escherichia coli Top10 (Sanggong Bioengineering (Shanghai) Co., Ltd.), it was placed with 0.1% ampicillin Cultured in penicillin LB liquid medium.
[0024] 3. Amplify by PCR, the reaction system is: Primer 1 (1μL), Primer 2 (1μL), 2×Taq Master Mix (10μL, Nanjing Novezan Biotechnology Co., Ltd.), E. coli culture broth (1μL) , DdH 2 O (7μL), total 20μL, amplification conditions: 94℃5min; 94℃35s, 52℃35s, 72℃1.5min, 30 cycles; 72℃10min, 4℃ storage.
[0025] Primer 1 (ZKE-primer 1): 5'- GGAATTC ATGATCAGGTGCATAGGAGTCAG-3'
[0026] Primer 2 (ZKE-primer 2): 5'-GCGGCCGCAGCAGAGACGGCTGTGGATA-3'
[0027] 4. The amplified products are detected by agarose gel electrophoresis. The results of agarose gel electrophoresis figure 1 As shown, an amplified product with a band of approximately 1515 bp was detected at 1500 bp of the standard molecular weight of the DNA, which is in line with the number of bases artificially designed for the Zika virus structural protein E gene fragment.
[0028] 5. Use the gel recovery kit of Beijing Quanshijin Company to recover the above PCR amplification products. After being digested with EcoRI and NotI, the recovered product was ligated with the pHMOXG-alpha-A vector digested with the same restriction endonuclease with T4 ligase. The ligation condition was 4℃ and the ligation time was 16h. The construction was successful. The plasmid was named pHMOXG-alpha-ZKE (or pHMOXG-α-ZKE). The upstream of the inserted target gene contains the co-expressed secretion signal peptide sequence, and the downstream contains the fusion expressed His tag sequence.
[0029] 6. The recombinant expression vector pHMOXG-alpha-ZKE was transformed into E. coli TOP10 competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) by heat shock method, and incubated in LB medium at 37°C at 160 rpm After culturing for 1 hour, the cells were collected by centrifugation, spread on LB solid medium containing 0.2% kanamycin, and cultured at 37°C for 16 hours for selection. Six transformants were randomly selected to inoculate 3 mL of LB liquid medium containing 0.1% kanamycin, and cultured at 37°C for 8 hours. Then use primer 1 and primer 2 to perform colony PCR, reaction system: bacterial solution 1μL, primer 1 and primer 2 are 1μL, 2×Taq Master Mix 10μL (Nanjing Novezan Biotechnology Co., Ltd.), ddH 2 O is 7 μL, and the total volume is 20 μL. The PCR amplification products are tested by agarose gel electrophoresis, and the test results are as follows figure 2 As shown, the designed Zika virus structural protein E gene fragment was detected at the DNA standard molecular weight of 1500 bp, and the target band was 1515 bp, which was in line with the artificially designed base number. That is, the positive Escherichia coli containing the recombinant expression vector pHMOXG-alpha-ZKE containing the Zika virus structural protein E gene fragment was successfully obtained.
[0030] Components of LB liquid medium containing 0.1% ampicillin: 1.0g/100mL ampicillin, 5g/1000mL yeast extract, 10g/1000mL tryptone, 10g/1000mL sodium chloride, NaOH adjusted to pH 7.0, 121°C, Autoclave for 20min.
[0031] LB solid medium containing 0.2% kanamycin Components: 2.0g/100mL kanamycin, 5g/1000mL yeast extract, 10g/1000mL tryptone, 10g/1000mL sodium chloride, 15g/1000mL agar powder , NaOH adjust the pH to 7.0, 121℃, 20min autoclave sterilization.
[0032] Components of LB liquid medium containing 0.1% kanamycin: 1.0g/100mL kanamycin, 5g/1000mL yeast extract, 10g/1000mL tryptone, 10g/1000mL sodium chloride, and NaOH to adjust pH to 7.0 , 121℃, 20min autoclave sterilization.
[0033] (2) Construction of recombinant Hansenula polymorpha capable of expressing Zika virus structural protein E
[0034] 2.1 Preparation of Hansenula polymorpha DL-1 competent cells
[0035] Hansenula polymorpha DL-1 was derived from ATCC (AT26012) and was obtained in March 2010.
[0036] 1. Pick a single colony of Hansenula polymorpha DL-1 from a fresh YPD plate and place it in 5mL YPD liquid medium and culture it at 37°C for 16 hours until the colony grows to saturation;
[0037] 2. Inoculate 1mL into 50mL YPD liquid medium, and cultivate to OD at 37℃ 600 =0.8-1.2 After centrifugation at 5000rpm for 8min, collect the cells;
[0038] 3. After adding 50mL pH8.0 TED (100mm Tris HCl and 50mm EDTA), place it in 37℃, 100rpm/min shaker for 30min, centrifuge at 4℃, 5000rpm/min for 8min to collect cells;
[0039] 4. After adding 50mL ice-cold 270mM sucrose to gently suspend the cells, centrifuge at 4℃, 5000rpm/min for 5min to collect the cells;
[0040] 5. After adding 25mL ice-cold 270mM sucrose to gently suspend the cells, collect the cells by centrifugation at 4°C and 5000rpm/min for 5min;
[0041] 6. Add 1 mL of ice-cold 270 mM sucrose to gently suspend the cells, and pack them in 80 μL/tube to prepare Hansenula polymorpha DL-1 competent cells.
[0042] 2.2 Linearization of recombinant expression vector pHMOXG-alpha-ZKE and construction of recombinant Hansenula polymorpha
[0043] Add 1 mL of E. coli broth containing the recombinant expression vector pHMOXG-alpha-ZKE to 50 mL of LB liquid medium, and incubate at 37°C and 200 rpm for 24 hours. The recombinant expression vector pHMOXG-alpha-ZKE was extracted with the plasmid extraction kit produced by Beijing Quanshijin Company.
[0044] The recombinant expression vector pHMOXG-alpha-ZKE was linearized with DraI, and the digestion reaction conditions were 37°C for 8 hours.
[0045] After linearization, the product is subjected to agarose gel electrophoresis, and the electrophoresis product is recovered with a gel recovery kit produced by Beijing Quanshijin, and dissolved in 20-30 μL of TE solution.
[0046] Electrotransformation: After gently mixing 20-30μL of linearized product with 80μL of Hansenula polymorpha DL-1 competent cells, immediately put it into the groove of the electric shock cup in an ice bath, ice bath for 5 minutes, use Bio-Rad The electroporator pulses the sample (pulse setting: 50μF, 200Ω, 1.5KV). After the shock is over, add ice-precooled 1mL YPD (containing 1mM MgCl). 2 ) Liquid culture medium, then transferred to a 2mL sterile centrifuge tube, cultured at 37°C, 100rpm for 1-2h, take 100-200μL and spread it on YPD (containing G418 at a final concentration of 300μg/mL) plate (YPDG for short), Incubate at 37°C for 48-96h and observe the appearance of transformants.
[0047] Randomly selected 20-30 transformants were inoculated into 5mL YPD liquid medium containing 300μg/mL G418, cultured at 37°C for 24h, centrifuged at 5000rpm/min for 5min to collect the cells, extract the genome, and perform PCR amplification with primer 1 and primer 2. Increase screening. The PCR amplification product was detected by agarose gel electrophoresis. The detection result detected the target band at the DNA standard molecular weight of 1500bp. The base number of the artificially designed Zika virus structural protein E gene fragment (1515bp) was obtained. The recombinant expression vector pHMOXG-alpha-ZKE of the recombinant expression vector of the structural protein E gene fragment of the card virus is a positive recombinant strain of Hansenula polymorpha.
[0048] 2.3 Expression of Zika virus structural protein E in recombinant Hansenula polymorpha
[0049] Inoculate the successfully constructed recombinant Hansenula polymorpha strain (the above-mentioned positive Hansenula polymorpha recombinant strain) into 5mL YPDG, culture at 37°C and 120rpm for 16h, then transfer the culture to 250mL YPD liquid medium to continue the culture After 24 hours, start induction (maintain the culture conditions), during which time, add methanol (inducer) every 12 hours until the final concentration reaches 0.5% (V/V). Induction is continued for 96 hours. During the induction period and the end of induction, the fermentation is collected by centrifugation at 4°C and 12000 rpm. The supernatant was concentrated by Millipore Amicon mLtra-15 (MWSO, 30000) to 20 mL for later use. Western blotting analysis was performed with His tag antibody. The test results are as follows image 3 As shown, the protein band was detected at the standard molecular weight of the protein at 55kDa, and the target protein was 55.6kDa, which was in line with the theoretical value. That is, a recombinant strain of Hansenula polymorpha expressing the structural protein E gene of Zika virus was successfully obtained. And as the induced expression time lasted from 48h to 72h, the expression of recombinant Zika virus structural protein E increased from 5.7mg/L to 8.6mg/L.
[0050] (3) Separation and purification of recombinant Zika virus structural protein E
[0051] Affinity chromatography pretreatment: add NaCl and NaH to the concentrated 20mL supernatant 2 PO 4 , Imidazole and Tris-HCl, the concentrations are 0.3M, 0.05M, 0.01M and 0.01M, respectively. The volume of the solution is fixed to 25mL. If the volume is insufficient, fill up with ultrapure water, and then filter with a 0.22μm filter.
[0052] 1) Ni column equilibration: Take 100 mL of equilibrium solution to equilibrate the Ni-NTA resin column.
[0053] 2) The pretreatment sample is processed by the above-mentioned balanced Ni column, and the flow-through is collected.
[0054] 3) Put 80mL washing solution (0.3M NaCl, 0.05M NaH 2 PO 4 , 0.01M Imidazole, 0.01MTris-HCl) to wash the protein that has not adsorbed on the Ni column, and collect 8 tubes in a row, each tube 10mL.
[0055] 4) Use 80mL eluent (0.3M NaCl, 0.05M NaH 2 PO 4 , 0.5M Imidazole, 0.01M Tris-HCl) to elute the target protein adsorbed on the Ni column, and continuously collect 16 tubes, 5 mL each, the target protein was detected in the third and fourth tubes of the eluate.
[0056] (4) Concentration and preservation of recombinant Zika virus structural protein E
[0057] The eluate containing the target protein (recombinant Zika virus structural protein E) purified by affinity chromatography was concentrated by centrifugation at 4000 rpm in an ultrafiltration tube with a pore size greater than 3.5 kD.
[0058] The concentrated protein was dialyzed in a dialysis bag in PBS buffer containing 5% (volume fraction) of glycerol for 12-16h (desalting).
[0059] Pass the dialysate through a 0.22μm filter, inject it into a sterile EP tube, and store it at -80°C for later use.
[0060] (5) Immunogenicity analysis of recombinant Zika virus structural protein E
[0061] (1) Animal immunity
[0062] Twenty-four female Balb/c mice aged 6-8 weeks were selected and randomly divided into 3 groups. Experimental group 1 immunogen was the expressed target protein (recombinant Zika virus structural protein E), and experimental group 2 immunogen was inactivated Cardiac virus, experimental group 3 was the negative control group (adjuvant), and received subcutaneous multi-point immunization. Experimental group 1 was immunized with 30μg of expressed recombinant Zika virus structural protein E and the same amount of Freund's complete adjuvant for the first immunization. After 2 weeks of the first immunization, the second immunization was performed with 15μg of expressed recombinant Zika virus structural protein. E and the same amount of Freund’s incomplete adjuvant were emulsified and immunized, followed by a booster immunization once a week, and a booster immunization twice. One week after the last booster immunization, blood was removed from the tail and centrifuged at 4500r/min for 10 minutes, and the serum was separated for use. . Experimental group 2 for the first immunization 1×10 5 PFU (Plaque-forming unit, PFU) inactivated Zika virus was emulsified with the same amount of Freund’s complete adjuvant and then immunized. The second immunization was performed 2 weeks after the first immunization, with 1×10 4 PFU inactivated Zika virus and the same amount of Freund’s incomplete adjuvant were emulsified and then immunized, followed by a booster immunization once a week, and a booster immunization twice. After the last booster immunization, the tail was removed and the blood was collected at 4500r/min. Centrifuge for 10 min, and separate the serum for later use. Experimental group 3 was immunized with 30μg Freund's complete adjuvant for the first immunization, and was immunized with 15μg Freund's incomplete adjuvant 2 weeks after the first immunization, followed by a booster immunization once a week, a booster immunization twice, and the last one One week after the booster immunization, the tail was removed and the blood was collected, centrifuged at 4500 r/min for 10 min, and the serum was separated for use.
[0063] (2) Determination of antibody titer
[0064] Using PRNT (Plaque reduction neutralization test, plaque reduction neutralization test), the experimental group 2 (mouse immunized with inactivated virus) mouse serum as positive serum, experimental group 3 (only immune adjuvant mouse group) small Mouse serum was used as a negative control serum. Hanks buffer was used as a blank control to analyze the antibody titer of the expressed recombinant Zika virus structural protein E immunized mice (experimental group 1). The results showed that the experimental group 1 was the expressed recombinant Zika virus structure The anti-Zika virus antibody titer in the protein E immunized mouse group reached 1:1280, indicating that the recombinant Zika virus structural protein E expressed in the present invention has certain immunogenicity.
