CN113403330A

CN113403330A - Modified new coronavirus S gene, recombinant plasmid and recombinant BCG vaccine constructed by same and application of recombinant plasmid and recombinant BCG vaccine

Info

Publication number: CN113403330A
Application number: CN202110807503.XA
Authority: CN
Inventors: 薛庆节; 于美美; 崔晏硕; 黄烁; 闫迎春; 陈廷
Original assignee: JINING MEDICAL UNIVERSITY
Current assignee: JINING MEDICAL UNIVERSITY
Priority date: 2021-04-30
Filing date: 2021-07-16
Publication date: 2021-09-17

Abstract

The invention discloses a modified new coronavirus SARS-CoV-2S protein, a recombinant plasmid constructed by the same, a recombinant BCG vaccine and application thereof, and belongs to the technical field of biological preparations, wherein the modified new coronavirus S gene complete sequence disclosed by the invention can be successfully expressed in an escherichia coli-tubercle bacillus shuttle plasmid pMV261 to obtain a recombinant plasmid pMVS by modifying a partial sequence of a gene promoter region and a partial sequence of a terminator region; and the recombinant plasmid pMVS is introduced into BCG through electrotransformation to obtain recombinant BCG (rBCG); the rBCG can successfully express the S protein, evoke the immune response of a human body, induce the generation of antibodies to prevent the invasion of viruses, and the constructed rBCG is a subunit vaccine, has more protection effect than the parental BCG, can prevent or treat new coronavirus and tubercle bacillus, and has huge social benefit.

Description

Modified new coronavirus S gene, recombinant plasmid and recombinant BCG vaccine constructed by same and application of recombinant plasmid and recombinant BCG vaccine

Technical Field

The invention relates to the technical field of biological preparations, in particular to the field of recombinant plasmids and recombinant BCG vaccine, and further relates to a modified novel coronavirus SARS-CoV-2S gene, a recombinant plasmid and a recombinant BCG vaccine constructed by the same and application thereof.

Background

The novel coronavirus (SARS-CoV-2) belongs to subgroup B in beta coronavirus, and its envelope has corolla-like protuberant S protein playing the most key role in virus attachment, fusion and entering into host cell. As the research of SARS-CoV-2S protein structure function and recombinant vaccine by molecular biologists proceeds, the research of S gene as a new SARS-CoV-2 recombinant vaccine attracts various fields of wide attention. Since the S protein of SARS-CoV-2 is exposed on the surface of the virus, it is involved in the recognition, binding and entry processes. Therefore, the method is used as the key for vaccine design and therapeutic antibody target development.

By 11/4/2021, the World Health Organization (WHO) published 273 vaccine development projects of COVID-19, 87 of which are currently in clinical trials, some vaccines have been approved for use in certain parts of the World. The existing 5 types of vaccines in China are applied and divided according to a technical route, and the 5 types of vaccines are divided into three types: the first is inactivated vaccine, including 3 types of inactivated vaccine produced by Chinese medicine Beijing company, Chinese Wuhan company and Beijing Kexing Zhongwei company; the adenovirus vector vaccine is a type 5 adenovirus vector vaccine produced by Tianjin conciseness; thirdly, recombinant protein vaccine, which is a recombinant novel coronavirus vaccine (CHO cell).

On one hand, the vaccines are special vaccines for the new coronavirus, and only can be used for the new coronavirus but cannot play a role in preventing and/or treating other viruses at the same time, on the other hand, the safety of the vaccines for children inoculation is to be tested, and if the vaccines are inoculated to children, relevant adverse reactions can occur. The new coronavirus vaccine mainly focuses on prevention and treatment of new coronavirus of children under six years old, and no corresponding vaccine exists at present in China, so that a huge market space exists.

Disclosure of Invention

In order to solve the problems, the invention aims to provide a modified novel coronavirus SARS-CoV-2S gene, a recombinant plasmid constructed by the same, a recombinant BCG (bacillus calmette-guerin) and application thereof, develops a vaccine aiming at the groups of teenagers below 18 years old and even children 6 years old, and recombines the vaccine with the BCG to achieve the aim of preventing and/or treating tuberculosis and diseases caused by the novel coronavirus simultaneously.

In order to achieve the purpose, the invention is realized by the following technical scheme:

a new coronavirus SARS-CoV-2S gene after modification is characterized in that partial sequences of a promoter region and partial sequences of a terminator region of the new coronavirus SARS-CoV-2S protein gene are modified so as to be expressed in shuttle plasmids; the base sequence of the modified SARS-CoV-2S gene of the new coronavirus is SEQ ID NO: 1.

the invention also comprises a recombinant plasmid pMVS for expressing the new coronavirus SARS-CoV-2S gene, the base sequence of the modified new coronavirus SARS-CoV-2S gene and the shuttle plasmid pMV261 of escherichia coli-tubercle bacillus are respectively cut by BamH I and Hind III, the cut sites are connected by T4 DNA ligase to obtain the recombinant plasmid pMVS; wherein the base sequence of the modified SARS-CoV-2S gene of the new coronavirus is SEQ ID NO: 1.

the invention also comprises a recombinant BCG vaccine rBCG, wherein the recombinant plasmid pMVS is introduced into BCG competent cells through electrotransformation to obtain the recombinant BCG vaccine rBCG.

The invention also includes the application of the recombinant plasmid pMVS for expressing the new coronavirus SARS-CoV-2S gene, the recombinant plasmid pMVS for expressing the new coronavirus SARS-CoV-2S gene is introduced into the existing vaccine to obtain the recombinant vaccine, the recombinant vaccine can also prevent or/and treat the new coronavirus on the premise of not influencing the application of the original vaccine, and the introduced existing vaccine can be used for preventing the germs such as typhoid bacillus, plague bacillus or Listeria, and the like.

The invention also includes the application of the rBCG in preparing the pharmaceutical preparation for preventing and/or treating pneumonia caused by new coronavirus SARS-CoV-2, and the application of the rBCG in preventing and/or treating tuberculosis is not influenced at the same time; the vaccine is especially suitable for teenagers under 18 years old, especially children under 6 years old, and has high safety and effectiveness.

Compared with the prior art, the invention has the following advantages:

the reformed new coronavirus S protein gene complete sequence of the invention can be successfully expressed in an escherichia coli-mycobacterium tuberculosis shuttle plasmid pMVS261 to obtain a recombinant plasmid pMVS by reforming a partial sequence of a gene promoter region and a partial sequence of a terminator region; and the recombinant plasmid pMVS is introduced into BCG through electrotransformation to obtain recombinant BCG (rBCG);

the recombinant BCG (rBCG) can successfully express the S protein, evoke the immune response of a human body, induce the generation of antibodies to prevent the invasion of viruses, and the constructed recombinant BCG (rBCG) is a subunit vaccine which has more protection effect than the parental BCG and can prevent or treat new coronavirus and tubercle bacillus; on the other hand, the BCG vaccine is an effective vaccine safe for infants, successfully expresses the new coronavirus S protein in the recombinant BCG vaccine, marks that the specific vaccine is provided for the prevention and treatment of new coronavirus of teenagers under 18 years, especially children under 6 years, and inevitably generates huge social benefit.

Drawings

FIG. 1 is a 1% agarose gel electrophoresis of the product of the PCR amplified SARS-CoV-2S gene;

FIG. 2 is a diagram of the products of the enzyme digestion of the S gene and pMVS;

FIG. 3 is a diagram showing the result of the PCR identification of S-pMVS;

FIG. 4 is a DNA sequencing map;

FIG. 5 is a Westernblot analysis chart of recombinant BCG expression product;

FIG. 6 is a 10X 100 times oil-scope observation bacterial morphology diagram at the time of the zilian acid-resistant staining;

FIG. 7 is a graph showing the results of the antibody titer of each group when the titer of the animal serum antibody is measured by ELISA;

FIG. 8 is a graph showing the results of measurement of IFN-. gamma.cytokine levels in serum;

FIG. 9 is a graph showing the results of measurement of IL-4 cytokine levels in serum;

FIG. 10 is a diagram showing the results of the proliferation of T cells by rBCG.

Reference numerals:

m1 DNA marker (DL5000), 1S gene PCR product, M2 DNA marker (DL10000), 2pMV261 enzyme digestion product, 3pMVS PCR product, 4 enzyme digestion pMVS, 5 protein molecular mass standard (Mr × 10)³) 6 recombinant bcg expressed S protein; 7 BCG vaccine control group, 8S gene enzyme digestion product.

Detailed Description

The invention aims to provide a modified novel coronavirus SARS-CoV-2S gene, a recombinant plasmid and a recombinant BCG vaccine constructed by the same and application thereof, and the invention is realized by the following technical scheme:

a new coronavirus SARS-CoV-2S gene after modification is characterized in that partial sequences of a promoter region and partial sequences of a terminator region of the new coronavirus SARS-CoV-2S protein gene are modified so as to be expressed in shuttle plasmids; the base sequence is SEQ ID NO: 1.

The invention also includes the application of recombinant plasmid pMVS expressing new coronavirus SARS-CoV-2S gene, the recombinant plasmid pMVS expressing new coronavirus SARS-CoV-2S gene is introduced into the existing vaccine to obtain recombinant vaccine, the recombinant vaccine can also prevent or/and treat new coronavirus on the premise of not influencing the use of the original vaccine; the existing vaccine which can be introduced is a vaccine for preventing pathogenic bacteria such as typhoid bacillus, plague bacillus or listeria.

The invention also includes the application of the rBCG in preparing the pharmaceutical preparation for preventing and/or treating pneumonia caused by the new coronavirus SARS-CoV-2, and simultaneously does not influence the application of preventing and treating tuberculosis; the vaccine is especially suitable for teenagers under 18 years old, especially children under 6 years old, and has high safety and effectiveness.

The invention is further described with reference to specific examples.

The reagents used for reverse transcription, namely '5X Prime Script IV cDNA Synthesis Mix' and 'Random 6 mers', and the manufacturers of the amplification system reagents 'Prime STAR GXL DNA Polymerase', 'Prime STAR GXL Buffer' and 'dNTP Mix' are Takara;

the manufacturers of S-BCD nionolonal antibody and Goatanti-Human IgG (H + L) are both Bioworld Technology, Inc, USA;

middlebrook 7H9 liquid medium was purchased from BD corporation, usa;

plasmid pMV261 used in the examples of the present invention was purchased from Biovector plasmid vector cell Gene Collection, but is not limited to this unit.

The RNA genome of SARS-CoV-2 virus is originated from the Huifeng of clinical laboratory of auxiliary hospital of Jining medical college, or purchased from the S gene of SARS-CoV-2 virus currently available in the market, such as Hanhengzheng Biotechnology (Shanghai) Co., Ltd.

Examples

In the invention, the main purpose of developing SARS-CoV-2 virus vaccine is to evoke human immune response and induce antibody to generate to prevent virus invasion, considering the characteristics of stability and easy transferability, etc., the pre-constructed recombinant BCG vaccine (rBCG) is subunit vaccine, and the rBCG has more protection effect than parental BCG, so the experiment adopts the mode of connecting the modified SARS-CoV-2S gene with BCG shuttle expression vector pMV261, and introduces recombinant plasmid into BCG competent cell by electric transformation to successfully express S protein, which has great significance for developing SARS-CoV-2 recombinant vaccine, especially for safe vaccine suitable for teenagers.

The S gene of the existing novel coronavirus is directly inserted into the shuttle expression plasmid pMV261, and then the expression of the S protein cannot be detected, so the technical difficulty lies in how to insert the S gene of the new coronavirus into the shuttle expression plasmid and successfully express the shuttle expression plasmid containing the S gene of the new coronavirus in the existing vaccine, and the prevention and treatment functions of diseases caused by the existing vaccine and the new coronavirus are realized simultaneously after the expression.

First, transform S gene complete sequence more beneficial to BCG expression

PCR amplification of S Gene

1.1 primer design

Upstream primer (S-F): CGG GGA TCC ATG TTC GTC TTC CTG GTC CTG, respectively;

downstream primer (S-R): GCG AAG CTT TTA GGT GTA ATG CAG CTT CAC, respectively;

the bold italics are the restriction sites, the restriction enzymes BamHI and HindIII respectively.

1.2 purification and recovery of amplified S Gene and product

Reverse transcription: 5XPrime Script IV cDNA Synthesis Mix 5. mu.1, Random 6mers (50. mu.M) 3. mu.1, RNA genome of SARS-CoV-2 virus: total RNA 1. mu.1 (25. mu.g/. mu.1), plus RNase (ribonuclease) -free ddH₂O to a final system of 20. mu.l; the reverse transcription procedure was: inactivating enzyme at 42 deg.C for 15min, inactivating enzyme at 80 deg.C for 10min, and cooling on ice;

PCR amplification was performed with the F primer and the R primer.

The amplification system was as follows: 0.2. mu.l of primer S-F, 0.2. mu.l of primer S-R, 1. mu.l of PrimeSTAR GXL DNA Polymerase, 10. mu.l of 5 XPimeSTAR GXL Buffer, 4. mu.l of dNTP mix, adding sterile water to make up to 50. mu.l, and mixing well. The reaction conditions are as follows: pre-denaturation at 98 deg.C for 4 min; performing co-amplification for 30 cycles at the denaturation temperature of 98 ℃, 10s, the annealing temperature of 60 ℃, 15s, the extension temperature of 68 ℃ and more than 4 min; further extension at 68 ℃ for 5 min. Separating by 1% agarose gel electrophoresis, purifying and recovering the amplification product, and operating according to the agarose gel DNA recovery kit purification and recovery instruction.

1.3 digestion, purification and recovery of PCR product

The PCR product S and the shuttle expression plasmid pMV261 were first digested with BamH I, 20. mu.l total, as follows: s and pMV261 were incubated overnight at 10. mu.l each, 1. mu.l BamHI, 2. mu.l 10 XKbuffer, supplemented to 20. mu.l with sterile water, 30 ℃. The resulting digested products were separated and recovered by electrophoresis on a 1% agarose gel, and the procedures were followed according to the agarose gel DNA recovery kit purification recovery instructions. The S and pMV261 BamH I purified products were each digested with HindIII, in a total of 20. mu.l, as follows: 10. mu.l each of the S and pMV261 BamH I cleavage products, 1. mu.l Hind III, 2. mu.l 10 XKbuffer, supplemented to 20. mu.l with sterile water, incubated at 37 ℃ overnight. The resulting digested products were separated and recovered by electrophoresis on a 1% agarose gel, and the procedures were followed according to the agarose gel DNA recovery kit purification recovery instructions.

Constructing recombinant plasmid and screening and identifying

The recovered product S and the shuttle expression plasmid pMV261 were ligated with T4 DNA ligase as follows: s34. mu.l, pMV 2616. mu.l, T4 DNA ligase 5. mu.l, T4 Buffer (10X) 5. mu.l, 16 ℃ overnight, ligation products were transferred into competent DH 5. alpha. transformation: the competent cells E.coli.DH5 alpha are placed on ice to be melted, 40 mu l of the competent cells E.coli.DH5 alpha are taken and mixed with 10 mu l S protein plasmids uniformly, ice bath is carried out for 30min, heat shock is carried out for 90s at 42 ℃, ice bath is carried out for 10min immediately, 800 mu l of LB liquid culture medium without antibiotics is added, the mixture is fully mixed, the mixture is placed in a shaking table at 37 ℃ and 160r/min for 1h, centrifugation is carried out at 5000r/min for 3min, supernatant liquid is sucked out for 700 mu l, the rest bacterial liquid is blown and suspended, the mixture is inoculated in LB solid culture medium (containing kanamycin (50 mu g/ml)), single colonies are picked and inoculated in LB liquid culture medium (containing kanamycin (50 mu g/ml)), the mixture is cultured at 37 ℃ and 160r/min overnight, and PCR identification and sequencing identification are carried out after plasmids are extracted by using a centrifugal column type plasmid miniextraction kit according to the instruction. The correctly identified recombinant SARS-CoV-2S plasmid was designated pMVS.

Thirdly, constructing recombinant BCG vaccine (rBCG)

3.1 preparation of BCG competent cells

Carrying out high-pressure steam sterilization on the Middlebrook 7H9 liquid culture medium at 110 ℃ for 15 minutes, selecting a BCG single colony to be inoculated into the Middlebrook 7H9 liquid culture medium containing kanamycin (50 mu g/mL) for liquid culture to obtain the Middlebrook 7H9 liquid culture medium containing BCG, carrying out culture at 37 ℃ and 100r/min until the culture reaches a logarithmic phase (the OD600/A600 value is 0.60), carrying out ice bath for 1.5H, then carrying out centrifugation at 4 ℃ and 5000r/min for 10min, adding 10% precooled glycerol with the volume of the culture solution of 1/10, then carrying out resuspension, carrying out centrifugation at 4 ℃ and 5000r/min for 10min, and then carrying out suspension precipitation with 10% glycerol. The washing is repeated for 3 times, and the BCG competent cells are obtained.

3.2 electrotransformation of BCG

The BCG competent cells were thawed on ice, and 10ul of pMVS was added to the BCG competent cells (about 9X 10)⁶pieces/mL), ice-cooled for 15min, and transferred to a 0.2cm pre-cooled electric rotor for electric transfer. The electrotransfer parameters are as follows: voltage: 2.5kv, electrotransfer time: 5.3 ms. After electroporation, 1ml of daily stock Peritose liquid medium was added quickly, the cells were resuspended slowly and transferred to EP tubes for overnight incubation at 37 ℃ and 100 r/min. The next dayCentrifuging the solution at 5000rpm for 10min, inoculating the precipitate to Middlebrook 7H10 solid culture medium containing 50ng/ml kanamycin, performing shake culture at 37 deg.C and 100r/min until vegetable-like colonies grow, and selecting single colony for liquid culture.

Fourth, data verification

Induced expression of rBCG and extraction of S protein

Measuring the OD595 value of the bacteria by using a spectrophotometer, when the OD595 value reaches 0.65, in the logarithmic growth phase, continuously inducing rBCG cultured to the logarithmic growth phase for 45 minutes at 45 ℃ for three days, centrifuging for 15 minutes at 4000rpm, collecting thalli, washing for 2 times by using PBS, adding a lysate (RIPA: PMSF ═ 1: 100), carrying out ultrasonic crushing after ice bath for 1.5 hours, and centrifuging at 4 ℃ after the completion, taking the supernatant, and carrying out SDS-PAGE electrophoresis;

functional verification of S protein

5.1 Western blot analysis of S proteins

Carrying out SDS-PAGE electrophoresis on the cultured recombinant BCG and BCG after ultrasonication, transferring bands in the gel to a PVDF membrane by a wet transfer method, and carrying out Western blot analysis after blocking, S-BCD nionolonal antibody (1) and HRP Goat Anti-Human IgG (H + L) incubation.

Wherein the wet transfer method transfers the bands in the gel to a PVDF membrane, and the specific operations of blocking, S-BCD nionolonal antibody (1) and HRP coat Anti-Human IgG (H + L) incubation are as follows:

after SDS-PAGE electrophoresis is finished, gel is stripped, and the PVDF membrane is immersed in methanol for 5 minutes and fully activated; then soaking the PVDF membrane, filter paper, sponge, a suction tube, a glass rod and the like in the prepared membrane transferring liquid for 2 hours; transferring the film for 1.5 hours at 150V in a refrigerator at 4 ℃; after the membrane is transferred, taking out the membrane, putting the membrane into 5% skim milk, and sealing the membrane for 1 hour at room temperature by shaking; TBST washing 3, 10 minutes each time; diluting the primary anti-S-BCD nionolonal antibody with 5% BSA (1: 1000), placing the membrane into a centrifuge tube containing the antibody, and incubating overnight in a shaker at 4 ℃; TBST washing for 3 times, 10 minutes each time; a second antibody, namely, a Goat Anti-Human IgG (H + L) labeled by horseradish peroxidase is diluted by 5% BSA (1: 5000), and is shaken for 1 hour in a shaking table at room temperature; TBST washing for 3 times, 10 minutes each time; preparing ECL working solution, exposing with X-ray film in a dark room, and observing the result;

PCR amplification of the S Gene

The SARS-CoV-2S gene (3822bp) was amplified by PCR. The amplification product was then analyzed by 1% agarose gel electrophoresis and was approximately 3800bp, as shown in FIG. 1, which is consistent with the expected results.

7. Enzyme digestion and ligation

The PCR product S gene and the BCG shuttle expression plasmid pMV261 were subjected to double digestion with BamHI and HindIII, respectively, to obtain digested fragments of 3.8kb and 4.5kb, respectively, as shown in FIG. 2, consistent with the expected results.

8. Construction and identification of recombinant plasmids

The target gene was amplified using S-pMVS as template and the forward (S-F) and reverse (S-R) primers, and the size of the PCR product was determined to be about 3800bp by 1% agarose gel electrophoresis, as shown in FIG. 3, which is essentially identical to theory.

DNA sequencing

The recombinant plasmid which is identified by PCR to be correct is sent to Shanghai engineering for sequencing, and the sequencing result is completely consistent with the modified SARS-CoV-2S gene sequence, as shown in figure 4, which shows that the target gene to be expressed is completely and correctly inserted.

Inducible expression of the S Gene

Carrying out wall breaking treatment on the recombinant BCG after thermal induction to extract S protein, taking a proper amount of protein sample to carry out SDS-PAGE electrophoresis, and carrying out Westernblot detection after electrotransformation. As shown in FIG. 5, the results showed that S protein was expressed positively in the recombinant plasmid BCG, and no band was observed in the BCG control group, indicating that the fusion of S protein in the recombinant plasmid BCG was successful.

11. Quick-dyeing of acid-fast

A proper amount of logarithmic growth phase rBCG (Middlebrook 7H9 liquid culture medium containing Kan + (50 mu g/mL) is respectively and uniformly smeared on a clean glass slide by using an inoculating loop, bacteria are fixed by quickly passing through the flame of an alcohol lamp, carbolic acid reddish solution is dripped, the glass slide is heated until the glass slide is exposed to steam, the flame is separated when the steam disappears, the operation is repeated for 5min, after the glass slide is cooled, the glass slide is washed by running water, 3% hydrochloric acid-alcohol are dripped for decoloration for 1min, the glass slide is washed by water, 0.3% alkaline methylene blue is dripped for dyeing for 30s, the water is washed, after the glass slide is dried, a neutral gum is sealed, the bacterial morphology is observed by using a 10 x 100 times oil microscope, photographing is carried out, and the result is recorded as shown in figure 6, which shows that the cultured BCG is successfully recombined.

Measurement of titer of animal serum antibody by ELISA method

The successfully constructed recombinant BCG is respectively injected into C57BL/6 mice with the age of 6 weeks subcutaneously, the injection dose is 100 mu L/mouse, the specific antibody of the mice is detected by an ELISA method, 5 mice in each group are controlled by PBS and empty carriers, the change of the antibody in the serum of the mice immunized by the recombinant BCG is measured, and the mice are immunized 1 time every 10 days for 3 times. Setting PBS as a control group; an empty vector control group; recombinant glandular BCG. Blood was drawn from the tail of the mice before the first injection, one day before the second and third injections, and every 10 days after the third injection, until 15 weeks after immunization; serum was isolated and specific antibodies in the serum were detected by ELISA.

In a 96-well plate, 0.25mL (1. mu.g/mL) of a carbonate buffer (0.05mol/L, pH 9.0) and 0.25mL (1. mu.g/mL) of a new coronavirus S protein solution were added to each well, the plate was coated overnight (4 ℃ C.), the well solution was discarded the next day, after washing with PBS buffer 3 times, 0.25mL of a freshly diluted enzyme-labeled antibody was added, incubation was performed at 37 ℃ for 1 hour, after washing thoroughly with PBS buffer 3 times, substrate TMB solution was added at 0.15mL37 ℃ for 0.5 hour, and 0.06mL of a sulfuric acid solution having a concentration of 1.85mol/L was added to terminate the reaction.

The S protein antibody titer of the new coronavirus SARS-CoV-2 in the serum of the experimental group mice is averagely as follows: 14.432, respectively; the S protein antibody titer of the new coronavirus SARS-CoV-2 in the serum of the BCG control group is averagely 3.056; the S protein antibody titer of the novel coronavirus SARS-CoV-2 in the serum of the PBS control group was 1.553 on average.

13. The double-antibody sandwich ELISA method is adopted to detect the levels of IL-4 and IFN-gamma cytokines in serum samples at 1, 3 and 5 weeks after the first immunization, and the experimental process is as follows:

the successfully constructed recombinant BCG is respectively injected into C57BL/6 mice of 6 weeks old subcutaneously with the injection dose of 100 mu L/mouse, the IL-4 and IFN-gamma cytokine levels in serum samples of 1, 3 and 5 weeks after injection are detected, a double-antibody sandwich ELISA method is adopted to detect specific antibodies, PBS and BCG groups are used as controls, 5 antibodies in each group are detected, and the specific method is carried out according to the specification of an ELISA kit of an eBioscience company:

1) add 100. mu.L of Capture antibody diluted with Coating Buffer (diluted 200-fold with 1 Xcoating Buffer) to each well of 96-well plate, and incubate overnight at 4 ℃ in the dark;

2) discarding the stock solution in the clean hole, adding 250 mu L of Wash Buffer into each hole, and washing for three times;

3) add 200. mu.L 1 XELISA/ELISAPOT Diluent (dilution) per well, stand 1h at room temperature for sealing;

4) preparing the standard substance for detection during the sample closing period, and adding dd H according to the instruction dosage required by the instruction₂O, after the mixture is acted for 30min at room temperature, the concentration of the mixture after being vibrated by a vortex vibrator is recorded as 500 pg/mL;

5) adding 250 mu L of Wash Buffer into each hole for washing once;

6) standard control group: 1-8 holes, sequentially adding 0, 100 and 100 muL of Diluent and 0 (adding 200 muL of standard stock solution) from top to bottom, sucking 100 muL of standard stock solution, and sequentially performing 2-fold gradient dilution;

7) adding 50 μ L/well of serum and 50 μ L of Diluent to the other wells, mixing, leaving a blank well, adding only 100 μ L of Diluent, and incubating at 4 deg.C for 8 hr;

8) washing for 3-5 times;

9) primary antibody preparation during incubation: DetectionIntfoods, 250-fold diluted with Diluent, 100. mu.L per well, and exposed for 1h at room temperature;

10) washing for 3-5 times;

11) during the incubation period, Avidin-HRP (secondary antibody is diluted 250 times with Diluent) is prepared, 100 mu L of the Avidin-HRP is added into each hole, the obtained product is sealed, and the obtained product is acted for 30min at room temperature;

12) washing for 6 times;

13) adding 100 μ L of 1 × TMB color developing agent into each well for color development, and acting at room temperature for 15 min;

14) adding 50 mu L of stop solution into each hole;

15) the reading is determined by setting 570nm in the microplate reader.

The results are shown in fig. 8-9 (ns > 0.1;. P < 0.05;. P <0.01 in the two figures), with the extension of immunity time, the BCG group, recombinant BCG group IFN-gamma, IL-4 cytokine level gradually increased, because IFN-gamma participates in the organism's cellular immune response, is the representative of Th1 type cytokine, IL-4 can stimulate the proliferation of B cell, participate in the humoral immune response, produce immunoglobulin, is the representative of Th2 type cytokine, can conclude, the recombinant BCG vaccine of the invention can induce the organism to produce Th1 type and Th2 type cytokines, and then produce balanced Th1 type cellular immune response and Th2 type humoral immune response.

14. Mouse lymphocyte proliferation assay

The successfully constructed recombinant BCG was subcutaneously injected into 6-week-old C57BL/6 mice at a dose of 100. mu.L/mouse, and splenic lymphocytes were isolated from the 6-week-old mice, and 3 mice were collected per group. The specific operation method comprises the following steps:

1) prepare 6 well cell culture plates and add 3mL PBS per well in the first row. And 3mL of serum-free and antibiotic-free RPMI-1640 culture medium is added to each hole in the second row, and the hole plate is marked with the serial number of the sample to be detected. Meanwhile, preparing 15mL centrifuge tubes, preparing 3 tubes for the same sample, wherein 5mL spleen lymphocyte separation solution is added into 1 tube, and 2 tubes are reserved and marked;

2) after the mice are killed, 75% alcohol is sprayed for disinfection, spleens are separated in a clean environment and immersed in PBS;

3) the spleen was removed from PBS, placed in a grinding mesh (200 mesh), and ground to a complete disruption in RPMI-1640 medium wells using 2mL of the bottom of the medical syringe handle;

4) sucking the suspension liquid of the spleen cells after grinding by a pipettor, filtering by a filter screen (200 meshes) again, filtering into a 50mL centrifuge tube, and centrifuging for 10min at 1500 rpm;

5) discarding the supernatant, resuspending the spleen cell pellet with 5mL RPMI-1640 culture medium, dripping into a 15mL centrifuge tube containing 6mL spleen lymphocyte separation fluid along the tube wall, and centrifuging at 1500rpm for 30 min;

6) centrifuging to obtain lymphocyte separation layer (white ring layer), sucking out white ring layer, transferring into new 15mL centrifuge tube, adding RPMI-1640 culture medium to 10mL, and centrifuging at 1500rpm for 10 min;

7) discarding the supernatant, adding 4mL of erythrocyte lysate, resuspending, gently shaking, mixing uniformly, and centrifuging at 1500rpm for 10 min;

8) discarding the supernatant, resuspending the cell pellet with 1mL of RPMI-1640 culture medium, and transferring to a 1.5mLEP centrifuge tube;

9) taking another EP tube, diluting the cells by 20 times by using PBS, and counting the cells;

10) selection of 3X 10⁶Carrying out flow analysis on each cell, adding the cell into a 1.5mL EP centrifuge tube, and preparing 2 parts; centrifuging at 3000rpm for 10min, and discarding the supernatant;

11) in the meantime, antibodies were prepared, each sample was resuspended with 100. mu.L of LPBS, APC anti-mouse CD3 Antibody (1. mu.L/test), FITC anti-mouse CD4 Antibody (1. mu.L/test), PE anti-mouse CD8a Antibody (1. mu.L/test), incubated at 4 ℃ for 30min, and 1 double negative control group and 3 single staining control groups were set;

12) after dyeing is finished, centrifuging at 3000rpm for 10min, discarding supernatant, then adding 1mL PBS for resuspension, washing twice, and centrifuging at 3000rpm for 5 min;

13) after resuspension in 1mL PBS, flow cytometry was performed.

CD3 is a molecular marker on the surface of mature T lymphocytes, CD8+ T cells have the main function of directly and specifically killing target cells, and CD4+ T cells have the main function of enhancing B cell-mediated humoral immune response, further generating antibodies and enhancing phagocyte-mediated anti-infection capacity; the experimental results are shown in FIG. 10, (ns > 0.1;. P <0.05), and it can be seen that the recombinant BCG vaccine as an immunogen can induce the organism to generate CD4+ T and CD8+ T cell immune responses, and the effect of the immune response is superior to that of the BCG vaccine.

Sequence listing

<110> Jining medical college

<120> modified novel coronavirus S gene, recombinant plasmid and recombinant BCG vaccine constructed by same and application thereof

<141> 2021-07-16

<150> 2021104833885

<151> 2021-04-30

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tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540

ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600

tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660

tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720

ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780

ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840

gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900

tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960

caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020

gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080

tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140

ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200

gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 1260

tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320

cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 1380

ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440

aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500

aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560

ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620

ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680

cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740

acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800

ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 1860

cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920

aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980

gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040

cctcggcggg cacgtagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100

gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160

agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220

tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280

acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa cacccaagaa 2340

gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400

aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460

ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520

cttggtgata ttgctgctag agacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580

ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640

acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700

caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760

aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc actttcttcc 2820

acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880

acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940

ctttcacgtc ttgacaaagt tgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000

cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060

atgttcgtct tcctggtcct gctgcctctg gtctcctcac agtgcgtcaa tctgacaact 3120

cggactcagc tgccacctgc ttatactaat agcttcacca gaggcgtgta ctatcctgac 3180

aaggtgttta gaagctccgt tttacattca actcaggact tgttcttacc tttcttttcc 3240

aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 3300

aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 3360

ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt 3420

aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 3480

ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 3540

tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 3600

ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 3660

tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 3720

tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 3780

ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 3840

ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 3900

gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 3960

tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 4020

caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 4080

gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 4140

tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 4200

ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 4260

gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 4320

tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 4380

cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 4440

ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 4500

aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 4560

aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 4620

ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 4680

ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 4740

cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 4800

acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 4860

ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 4920

cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 4980

aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 5040

gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 5100

cctcggcggg cacgtagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 5160

gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 5220

agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 5280

tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 5340

acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa cacccaagaa 5400

gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 5460

aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 5520

ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 5580

cttggtgata ttgctgctag agacctcatt tgtgcacaaa agtttaacgg ccttactgtt 5640

ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 5700

acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 5760

caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 5820

aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc actttcttcc 5880

acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 5940

acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 6000

ctttcacgtc ttgacaaagt tgaggctgaa gtgcaaattg ataggttgat cacaggcaga 6060

cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 6120

tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 6180

gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 6240

gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 6300

atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 6360

cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca 6420

tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 6480

ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcatacc 6540

tcccctgacg tggatctggg cgacatcagc ggcatcaatg cctccgtggt gaacatccag 6600

aaggagatcg accgcctgaa cgaggtggct aagaatctga acgagagcct gatcgacctc 6660

caggagctgg gcaagtatga gcagtacatc aagtggccct ggtacatctg gctgggcttc 6720

atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgtat gacatcctgc 6780

tgttcttgcc tgaagggctg ctgtagctgt ggctcctgct gtaagtttga cgaggatgac 6840

tctgaacctg tgctgaaggg cgtgaagctg cattacacct aa 6882

Claims

1. A modified new coronavirus SARS-CoV-2S gene is characterized in that: transforming partial sequence of promoter region and partial sequence of terminator region of new coronavirus SARS-CoV-2S protein gene to make it be expressed in shuttle plasmid; the base sequence of the modified SARS-CoV-2S gene of the new coronavirus is SEQ ID NO: 1.

2. a recombinant plasmid pMVS for expressing new coronavirus SARS-CoV-2S gene is characterized in that the base sequence of the modified new coronavirus SARS-CoV-2S gene and Escherichia coli-tubercle bacillus shuttle plasmid pMV261 are respectively cut by BamH I and Hind III, and the cut sites are connected by T4 DNA ligase to obtain recombinant plasmid pMVS;

wherein the base sequence of the modified SARS-CoV-2S gene of the new coronavirus is SEQ ID NO: 1.

3. a recombinant BCG vaccine rBCG, which is characterized in that: the recombinant plasmid pMVS expressing the new coronavirus SARS-CoV-2S gene of claim 2 is introduced into BCG competent cells by electrotransformation to obtain recombinant BCG vaccine rBCG.

4. The use of the recombinant plasmid pMVS expressing the SARS-CoV-2S gene of the novel coronavirus as set forth in claim 2, wherein: introducing into the existing vaccine to obtain the recombinant vaccine.

5. The use of recombinant BCG rBCG according to claim 3, characterized in that: the application in preparing medicine preparation for preventing and/or treating pneumonia caused by new coronavirus SARS-CoV-2.