CN104857511B - Thinner for vaccine containing panaxoside - Google Patents

Abstract

The invention discloses a kind of thinner for vaccine containing panaxoside, and it is made up of the composition of following weight content：0.004%~0.012% stem and leaf of Radix Ginseng saponin or panaxoside, 0.001%~0.01% thimerosal, surplus is physiological saline.The immune effect of vaccine can be increased substantially with containing immune animal after stem and leaf of Radix Ginseng saponin (GSLS) or the thinner for vaccine of panaxoside (GS) and thimerosal dilution vaccine；One of main component of diluent thimerosal has preservative efficacy, therefore prevents product mildew without additionally adding preservative.

Description

Vaccine Diluent Containing Ginsenosides

technical field

The invention relates to a preparation method and application of a vaccine diluent containing ginsenoside.

Background technique

my country is the largest breeding country in the world, with about 1 billion (heads) of pigs, cattle, and sheep slaughtered each year (1). In recent years, due to the ravages of animal infectious diseases, a large number of livestock and poultry have died, which has seriously endangered the healthy development of the breeding industry. After the level of large-scale breeding is improved, the disease is more likely to spread, and the task of animal disease prevention and control is very arduous. In order to reduce the morbidity and mortality of animals, the state stipulates that certain animal infectious diseases must be immunized against vaccines. However, in recent years, the phenomenon of vaccine immunization failure in the breeding industry has occurred from time to time. After the animals are immunized and vaccinated, no sufficient level of antibodies can be detected, which cannot prevent the outbreak of the disease and cause serious economic losses to the farmers. For example, Zhu Anmin et al. detected serum antibodies of 95 pigs after the second injection of FMD vaccine and showed that the qualified rate of immunity was only 64% (2); Chen Biao et al. 41.4% of the pigs achieved acceptable antibody levels (3). Therefore, it is of great significance to improve the immune effect of vaccines and effectively prevent the occurrence of infectious diseases in the aquaculture industry.

Adding adjuvants to animal vaccines is one of the measures to improve the immune effect of vaccines. At present, the vaccines injected into animals mainly include attenuated vaccines and inactivated vaccines. Improving the immune effect of vaccines by adding adjuvants is mainly seen in inactivated vaccines, such as inactivated foot-and-mouth disease virus oil adjuvant vaccines. Freeze-dried attenuated vaccines are widely used in the breeding industry due to the long duration of antibodies after immunization, such as porcine pseudorabies attenuated vaccine, infectious bursal disease attenuated freeze-dried vaccine, swine fever freeze-dried vaccine, Newcastle disease IV series attenuated vaccine Wait. This type of vaccine needs to be diluted with a diluent before it can be used for immunization of animals. Commonly used vaccine diluents include aluminum hydroxide aluminum gel saline solution and normal saline. The disadvantage of the aluminum colloid solution is that it only improves the immune response of the cells, the local irritation reaction at the injection site is large, and the frozen storage is easy to destroy the stability of the aluminum colloid and make it lose the adjuvant function; the normal saline diluent has no immune enhancement effect.

Thimerosal, also known as sodium thimerosalate, is a mercury-containing organic compound that has long been widely used as a preservative for biological products and pharmaceutical preparations, including many vaccines, to inhibit the growth of microorganisms in the drug. That is, the only known use of thimerosal in vaccines is for preservation.

The references involved are as follows:

(1) "China Agricultural Yearbook 2013", pages 241-242, 257;

(2) Zhu Anmin, Wang Fangfang, Yao Zhansheng, Liu Weifeng, Swine Foot-and-Mouth Disease Vaccine Tracking Antibody Detection Test and Suggestion after Immunization, Animal and Poultry Industry, 2014, (307): 5-7;

(3) Chen Biao, Cai Kuojun, Li Aiqiao, Shi Yuanxiang, Chen Faxi, Wang Liuhe, Li Jianling, Zuo Yong, Monitoring and Analysis of Immune Efficacy of Different Vaccines for Type O Foot-and-Mouth Disease in Urumqi City, Contemporary Animal Husbandry, 2014, (5): 43- 45).

Contents of the invention

The technical problem to be solved by the present invention is to provide a vaccine diluent containing ginsenoside, which can overcome the shortcomings of existing vaccine diluents such as weak effect on cellular immune response and no immune enhancement effect.

In order to solve the above-mentioned technical problems, the present invention provides a vaccine diluent containing ginsenosides, which is composed of the following components by weight: 0.004% to 0.012% ginsenosides (GSLS) or ginsenosides (GS), 0.001% ~0.01% thimerosal (thimerosal sodium salt), the balance is normal saline.

The preparation method of the present invention is: dissolving the ginseng stem and leaf saponins (GSLS) or ginsenosides (GS) and thimerosal in physiological saline, and filtering through a 0.22 μm filter membrane to sterilize, and the resulting sterile solution is ginsenoside-containing vaccine diluent.

As an improvement of the ginsenoside-containing vaccine diluent of the present invention: the pH value of the vaccine diluent is 6-8; that is, the pH produced after the solution of the present invention is prepared is about 7. Ginsenosides and thimerosal are relatively stable and highly active in this neutral solution.

As a further improvement of the ginsenoside-containing vaccine diluent of the present invention: the content of ginsenoside or ginsenoside is 0.006%, and the content of thimerosal is 0.01%.

Remarks: Physiological saline is an aqueous solution containing 0.85-0.9% sodium chloride, which is isotonic with mammalian plasma; this is a conventional technique.

Thimerosal sodium salt (Merthiolate sodium, C ₉ H ₉ HgNaO ₂ S, relative molecular weight 404.30) molecular structure formula is as follows:

The diluent of the present invention can be used to dilute the vaccine and then inject animals, which can greatly improve the vaccine-induced cellular and humoral immune responses, reduce local irritation at the injection site, and can be frozen to prolong the storage period of the diluent. Since thimerosal, one of the main components of the diluent, has antiseptic effect, there is no need to add additional preservatives to prevent the product from growing moldy when the vaccine diluent is prepared industrially.

In summary, the present invention solves the following technical problems: (1) the existing aluminum glue vaccine diluent mainly promotes the humoral immune response, but the effect on the cellular immune response is weak; (2) the injection caused by the existing aluminum glue diluent The local irritation reaction of the site is large; (3) the existing aluminum gel diluent vaccine cannot be frozen; that is, the property is unstable; (4) the existing diluent has no immune enhancing effect.

The beneficial effect of the present invention is: the joint application of ginseng stem and leaf saponin (GSLS) or ginsenoside (GS) and thimerosal exerts a stronger immune enhancing effect than that of each of them alone. Immunizing animals after diluting the vaccine with a vaccine diluent containing ginsenosides (GSLS) or ginsenosides (GS) and thimerosal can greatly improve the immune effect of the vaccine; one of the main components of the diluent, thimerosal, has antiseptic properties and is prepared in industry Vaccine diluents do not require additional preservatives to prevent mold growth.

Description of drawings

The specific implementation manners of the present invention will be described in further detail below in conjunction with the accompanying drawings.

Figure 1 is a comparison chart of the effects of different diluent formulations on the antibody levels of mice immunized with attenuated pseudorabies vaccine (different superscript letters indicate significant differences between groups, P<0.05);

Figure 2 is a comparison chart of the synergistic effect of ginsenoside (GS) and thimerosal (MS) to promote antibody production;

Fig. 3 is the comparison chart of diluent (GSLS-MS) etc. on the level of gB antibody (blocking rate) after the mice are immunized with PRV attenuated vaccine;

Fig. 4 is a comparison chart of the influence of diluent (GSLS-MS) etc. on the level of IgG1 after the mouse is immunized with PRV attenuated vaccine;

Fig. 5 is a comparison chart of the influence of diluent (GSLS-MS) etc. on the level of IgG2a after the mouse is immunized with PRV attenuated vaccine;

Fig. 6 is that diluent (GSLS-MS) has significantly improved the IFN-γ level of mouse serum;

Figure 7 is that the diluent (GSLS-MS) significantly enhanced the killing activity of NK cells;

Fig. 8 is the effect of diluent (GSLS-MS) on mouse spleen lymphocyte proliferation response;

Fig. 9 is the effect of diluent (GSLS-MS) on mouse splenocytes producing cytokines;

Fig. 10 is the impact of diluent (GSLS-MS) on the challenge protection rate after the mouse is immunized with PRV attenuated vaccine;

Fig. 11 is the effect of diluent (GSLS-MS) on the antibody level after pig immunization PRV attenuated vaccine;

Fig. 12 is the serum antibody level after diluting pseudorabies vaccine with different diluents;

Figure 13 shows the effect of diluents containing different doses of MS on serum antibody levels.

Detailed ways

Remarks: In the following cases, the percentages related to the concentration/content of ginseng stem and leaf saponins (GSLS) or ginsenosides (GS) and thimerosal all refer to weight %.

Embodiment 1: Screening research of vaccine diluent formulation

1. Materials and methods

1. Reagents

Thimerosal sodium salt (MS), batch number: F20030210, purchased from China Pharmaceutical Group Shanghai Chemical Reagent Company; sodium chloride injection, batch number: B13020601, purchased from Zhejiang Guojing Pharmaceutical Co., Ltd.; porcine pseudorabies virus gB antibody detection kit , batch number: 09732-FC905, purchased from IDEXX company in the United States; ginseng stem and leaf saponins (GSLS), batch number: HJ140301, total saponin content 80.23%, containing Rg1 6.0%, Re 16.36%, Rbl 2.4%, Rb2 3.8%, Rf 0.1 %, Rc 3.70% and Rd9%, purchased from Jilin Hongjiu Pharmaceutical Co., Ltd.

2. Preparation of vaccine diluent containing ginsenosides and thimerosal

Preparation of thimerosal sodium salt solution: Weigh a certain amount of thimerosal sodium salt and fully dissolve it in physiological saline for injection to prepare a solution with a concentration of 0.002% (% by weight), and store it at 4°C for future use.

Preparation of ginseng stem and leaf saponin solution: take a certain amount of ginseng stem and leaf saponin and fully dissolve it in physiological saline for injection, and make solutions containing ginseng stem and leaf saponin concentrations (weight concentration) of 0.008%, 0.012% and 0.016% respectively , stored at 4°C for later use.

The preparation of 8 kinds of vaccine dilutions: with the above-mentioned physiological saline, ginseng stem and leaf saponin solution and thimerosal sodium salt solution as the mother solution, prepare 8 kinds of dilutions: the ginseng stem and leaf saponin content (weight %) in dilution 1～4 is respectively 0, 0.004%, 0.006% and 0.008%; dilutions 5 to 8 all contain 0.001% sodium thimerosal, but the contents of ginseng stem and leaf saponins are 0, 0.004%, 0.006% and 0.008%, respectively. All 8 solutions were sterilized by filtration with 0.22 μm filter membrane.

3. Vaccines

Live pseudorabies vaccine (Bartha-K61 strain, batch number: 130901) was purchased from Hangzhou Jianliang Veterinary Biological Products Co., Ltd.

4. Animal Grouping and Immunization

Fifty-four 4-week-old ICR female mice were randomly divided into 9 groups, 6 in each group. Dilute the attenuated pseudorabies vaccine with the 8 dilutions obtained in step 2 respectively. According to Table 1 grouping, mice were immunized with vaccines. In addition to the injection of normal saline in group 1, each mouse was injected with diluted pseudorabies attenuated vaccine (the half infectious dose of virus tissue (TCID50) was 600, the volume was 100 μL), and the mice were immunized twice with an interval of 2 weeks.

Table 1. Grouping and treatment of mice

5. Sampling

At the 2nd and 3rd week after the second immunization, about 0.4ml of orbital blood was collected, centrifuged at 4000rpm for 10 minutes, the serum was separated, and stored at -80°C.

6. Antibody detection

Use the porcine pseudorabies virus gB antibody detection kit to detect the serum pseudorabies gB antibody level, and the specific steps are as follows:

(1) Return all reagents taken out of the refrigerator to room temperature.

(2) Add 100 μL of 2-fold diluted negative control, positive control and serum samples to the antigen-coated plate.

(3) Incubate at 18-26°C for 60 minutes.

(4) Wash the micropores 3 to 5 times with the washing solution.

(5) Add 100 μL enzyme-labeled antibody to each well and incubate at 18-26°C for 20 minutes.

(6) Repeat step 4.

(7) Add 100 μL of TMB substrate to each well, and incubate at 18-26° C. for 15 minutes.

(8) Add 50 μL of stop solution to each well to stop the reaction, and read the absorbance at 650 nm.

7. Statistical analysis of antibody levels with mean ± standard deviation (Mean ± S.D.), using SPSS (17.0) statistical software in one-way ANOVA (one-way ANOVA), and Duncan method for multiple comparisons between groups, P< 0.05 means the difference is significant, P<0.01 means the difference is extremely significant, Microsoft Office Excel 2007 software for drawing.

2. The results are shown in Figure 1.

As can be seen from Figure 1, the serum antibody level obtained by diluting PRV attenuated vaccine immunized animals with 0.006% GSLS solution as diluent (group 4) was significantly higher than that of normal saline diluent (group 2) (P<0.05). The 0.001% thimerosal solution (group 6) had a significant immune enhancing effect (P<0.05) than the normal saline diluent (group 2). Thimerosal (MS) (0.001%) and GSLS (0.006%) combined to produce immune enhancement was significantly higher than that of the two alone (P <0.05).

To sum up, the present invention not only finds that thimerosal has the effect of immune adjuvant, but also finds that the combined use of thimerosal and GSLS has a stronger immune synergistic effect than when they are used alone.

Example 2: Research on the synergistic adjuvant effect of ginsenosides and thimerosal

1. Materials and methods

1. Reagent thimerosal sodium salt (MS), batch number: F20030210, purchased from China Pharmaceutical Group Shanghai Chemical Reagent Company; sodium chloride injection, batch number: B13020601, purchased from Zhejiang Guojing Pharmaceutical Co., Ltd.; porcine pseudorabies virus gB antibody Detection kit, batch number: 09732-FC905, purchased from IDEXX company in the United States; ginsenosides (GS), total saponins greater than 90%; containing Rb1 (18.0%), Rb2 (9.5%), Rc (10.0%), Rd (7.6 %), Re (8.7%) and Rg1 (3.5%).

2. The preparation of thimerosal sodium salt (MS) solution containing ginsenoside and thimerosal solution: take a certain amount of thimerosal sodium salt and fully dissolve it in normal saline, adjust the solution concentration to 0.02% (weight %), and store at 4°C spare.

Preparation of ginsenoside (GS) solution: take a certain amount of ginsenoside and fully dissolve it in normal saline, adjust the concentration of the solution to 0.012% (weight %), and store it at 4°C for later use.

The preparation of vaccine diluent (GS-MS): above-mentioned ginsenoside solution (0.012%) and thimerosal sodium salt solution (0.02%) are mixed by equal weight, get vaccine diluent, containing ginsenoside (0.006%) and thimerosal sodium salt ( 0.01%), and the solution was sterilized by filtration through a 0.22 μm membrane filter.

3. Vaccine Live pseudorabies vaccine (Bartha-K61 strain, batch number: 130901) was purchased from Hangzhou Jianliang Veterinary Biological Products Co., Ltd.

4. Animal grouping and immunization Thirty 4-week-old ICR female mice were randomly divided into 5 groups, 6 in each group. Dilute the attenuated pseudorabies vaccine with physiological saline, ginsenoside solution, thimerosal solution, ginsenoside-thimerosal solution (GS-MS) respectively. According to Table 2, the mice were immunized and injected with the vaccine. In addition to the injection of normal saline in group 1, each mouse was injected with diluted immune pseudorabies attenuated vaccine (the half infectious dose of virus tissue (TCID50) was 600, and the volume was 100 μL), and the mice were immunized twice with an interval of 2 weeks.

Table 2. Grouping and treatment of mice

group mouse GS dose (μg) Thimerosal dosage (μg) Pseudorabies Attenuated Vaccine 1 6 0 0 - 2 6 0 0 + 3 6 6 (0.006%) 0 + 4 6 0 10 (0.01%) + 5 6 6 (0.006%) 10 (0.01%) +

5. Sampling Take about 0.4ml of orbital blood at 2 and 3 weeks after the second immunization, centrifuge at 4000rpm for 10 minutes, separate the serum, and store at -80°C.

6. Antibody detection Use the porcine pseudorabies virus gB antibody detection kit to detect the serum pseudorabies gB antibody level, the specific steps are as follows:

(1) Return all reagents taken out of the refrigerator to room temperature.

(2) Add 100 μL of 2-fold diluted negative control, positive control and serum samples to the antigen-coated plate.

(3) Incubate at 18-26°C for 60 minutes.

(4) Wash the micropores 3 to 5 times with the washing solution.

(5) Add 100 μL enzyme-labeled antibody to each well and incubate at 18-26°C for 20 minutes.

(6) Repeat step 4.

(7) Add 100 μL of TMB substrate to each well, and incubate at 18-26° C. for 15 minutes.

(8) Add 50 μL of stop solution to each well to stop the reaction, and read the absorbance at 650 nm.

7. Statistical analysis of antibody levels with mean ± standard deviation (Mean ± S.D.), using SPSS (17.0) statistical software in one-way ANOVA (one-way ANOVA), and Duncan method for multiple comparisons between groups, P< 0.05 means the difference is significant, P<0.01 means the difference is extremely significant, Microsoft Office Excel 2007 software for drawing.

2. The results are shown in Figure 2.

As can be seen from Figure 2, the immunoenhancing effect produced by combined use of thimerosal (MS) (0.01%) and ginsenoside (GS) (0.006%) is significantly higher than that of the two alone (P<0.05), indicating that thimerosal and ginsenoside GS has a synergistic immune enhancing effect.

Example 3: Vaccine diluents enhance serum antibody levels

1. Materials and methods

1. Reagent thimerosal sodium salt (MS), batch number: F20030210, purchased from China Pharmaceutical Group Shanghai Chemical Reagent Company; sodium chloride injection, batch number: B13020601, purchased from Zhejiang Guojing Pharmaceutical Co., Ltd.; porcine pseudorabies virus gB antibody Detection kit, batch number: 09732-FC905, purchased from IDEXX, USA; ginseng stem and leaf saponins (GSLS): batch number: HJ140301, total saponin content 80.23%, containing Rg1 6.0%, Re 16.36%, Rbl 2.4%, Rb2 3.8% , Rf0.1%, Rc 3.70% and Rd 9%, purchased from Jilin Hongjiu Pharmaceutical Co., Ltd. HRP-labeled goat anti-mouse IgG1 and IgG2a antibodies, lot number: B13020601, were purchased from Santa Cruz Biotechnology Co., Ltd.

2. Preparation of vaccine diluent First prepare 0.002% (weight %) thimerosal normal saline solution as solution A; then prepare 0.012% (weight %) GSLS normal saline solution as solution B. Combine the two liquids A and B in equal weight, and filter the obtained liquid with a 0.22 μm filter membrane to obtain the vaccine dilution.

3. Vaccine Attenuated pseudorabies vaccine, Bartha-K61 strain, batch number: 130901, purchased from Hangzhou Jianliang Veterinary Biological Products Co., Ltd.

4. Animal grouping and immunization 18 ICR mice were randomly divided into 3 groups, 6 in each group. Group 1: dilute vaccine with vaccine diluent, immunize mice by intramuscular injection, the dose of each mouse is 1800TCID ₅₀ ; Group 2: dilute vaccine with normal saline, immunization method and vaccine dosage are the same as group 1; Group 3: use and above-mentioned Both groups injected normal saline to the mice in the same way. Each mouse was injected twice, each injection volume was 0.1 ml, with an interval of 2 weeks.

5. Sampling: At 2, 3, 4, 5, and 6 weeks after the second immunization, blood was collected from the orbit, and the serum was separated to detect antibodies and antibody sublevels.

6. Detection of serum antibodies and their subtypes Use the porcine pseudorabies virus gB antibody detection kit from the American IDEXX company to detect the serum PRV gB antibody level by ELISA antibody blocking method (see Example 1 for specific steps).

Serum PRV gB antibody IgG2a, IgG1 subclass levels were detected using the antigen-coated plate, washing solution and TMB substrate solution of the porcine pseudorabies virus gB antibody detection kit from IDEXX company. Specific steps are as follows:

(1) Add 100 μL of 2-fold diluted sample and negative serum to each well.

(2) Incubate at 18-26°C for 60 minutes.

(3) Wash the microwells 3-5 times with approximately 300 μL of washing solution per well.

(4) Add 1:1000 dilution of HRP-labeled goat anti-mouse IgG2a or IgG1 antibody, 100 μL per well, and incubate at 18-26°C for 60 minutes.

(5) Repeat step (3).

(6) Add 100 μL of TMB substrate solution to each well and incubate at 18-26° C. for 15 minutes.

(7) Add 50 μL of 2M H2SO4 to each well to terminate the reaction, measure and record the absorbance at 450 nm.

7. Statistical analysis Antibody test results are expressed as mean ± standard deviation (Mean ± S.D.). One-way ANOVA in SPSS (l7.0) statistical software was used, and Duncan method was used for multiple comparisons between groups, and Microsoft Office Excel 2007 software was used for drawing.

2. The results are shown in Figures 3-5.

Figures 3 to 5 respectively represent the levels of serum gB antibody, IgG1 and IgG2a in the three groups of mice after immunization. Among the three groups, the antibody level and its subtypes in the vaccine diluent group were significantly higher than those in the normal saline group (P<0.05).

Example 4: Vaccine diluents can enhance serum interferon gamma levels and activity of cytotoxic lymphocytes

1. Materials and methods

1. Reagent thimerosal sodium salt (MS), batch number: F20030210, purchased from China Pharmaceutical Group Shanghai Chemical Reagent Company; sodium chloride injection, batch number: B13020601, purchased from Zhejiang Guojing Pharmaceutical Co., Ltd.; porcine pseudorabies virus gB antibody Detection kit, batch number: 09732-FC905, purchased from IDEXX, USA; ginseng stem and leaf saponins (GSLS): batch number: HJ140301, total saponin content 80.23%, containing Rg1 6.0%, Re 16.36%, Rbl 2.4%, Rb2 3.8% , Rf0.1%, Rc 3.70% and Rd 9%, purchased from Jilin Hongjiu Pharmaceutical Co., Ltd. Mouse interferon IFN-γELISA detection kit, batch number: 228040611, purchased from Hangzhou Lianke Biotechnology Co., Ltd.; mouse lymphoma cells (YAC-1), a gift from the Institute of Immunology, Zhejiang University School of Medicine; RPMI 1640, batch number: NZF1156 was purchased from Thermo Fisher Biochemical Products (Beijing) Co., Ltd.; premium fetal bovine serum (FBS) (endotoxin <10EU/ml) was purchased from Hyclone Company of the United States; red blood cell lysate, lot number: 20130918, was purchased from Beijing Suo Laibao Biotechnology Co., Ltd.

2. Preparation of vaccine diluent First prepare 0.002% (weight %) thimerosal normal saline solution as solution A; then prepare 0.012% (weight %) GSLS normal saline solution as solution B. Combine the two liquids A and B in equal weight, and filter the obtained liquid with a 0.22 μm filter membrane to obtain the vaccine dilution.

3. Vaccine Attenuated pseudorabies vaccine, Bartha-K61 strain, batch number: 130901, purchased from Hangzhou Jianliang Veterinary Biological Products Co., Ltd.

4. Animal grouping and immunization 18 ICR mice were randomly divided into 3 groups, 6 in each group. Group 1: dilute vaccine with vaccine diluent, immunize mice by intramuscular injection, the dose of each mouse is 1800TCID ₅₀ ; Group 2: dilute vaccine with normal saline, immunization method and vaccine dosage are the same as group 1; Group 3: use and above-mentioned Both groups injected normal saline to the mice in the same way. Each mouse was injected twice, each injection volume was 0.1 ml, with an interval of 2 weeks.

5. Sampling 24 hours after immunization, blood was collected from the eyes of the mice to prepare serum for the detection of IFN-γ content; the spleen was isolated to prepare splenocytes, and the activity of NK cells was detected by MTT method.

6. Detection of serum interferon Using the mouse IFN-γ Sunny ELISA detection kit, the level of serum IFN-γ was detected by the double antibody sandwich ELISA method. Specific steps are as follows:

(1) All reagents taken out of the refrigerator should be brought back to room temperature before use.

(2) Wash the plate twice with 300 μl washing solution per well.

(3) Add 50 μl of detection buffer, 50 μl of sample (or standard or control) and 50 μl of detection antibody to each well in sequence.

(4) Seal the plate with a sealing film, shake at 100 times/min, and incubate at room temperature for 2 hours.

(5) Wash the plate 6 times with 300 μl washing solution.

(6) Add 100 μL horseradish peroxidase-labeled streptavidin to each well.

(7) Seal the plate with a sealing film, shake at 100 times/min, and incubate at room temperature for 45 minutes.

(8) Repeat step (5).

(9) Add 100 μl chromogenic substrate TMB to each well, protect from light, and incubate at room temperature for 10-30 minutes.

(10) Add 100 μl of stop solution to each well and mix well.

(11) Within 30 minutes, measure the OD value with a microplate reader at a wavelength of 450nm, and the reference wavelength is 570nm.

(12) Calculate the content of cytokines with the standard curve.

7. NK cell killing activity is detected according to the following steps:

(1) Preparation of effector cells (mouse splenic lymphocytes) Separation of mouse splenic lymphocytes, and then use erythrocyte lysate to remove red blood cells, then wash the cells twice with PBS, and suspend the cells in complete medium (modified RPMI 1640+10 %FBS), the cell suspension was divided into two tubes, and the cell concentration was adjusted to 1×10 ⁷ /ml and 5×10 ⁶ /ml respectively.

(2) Preparation of target cells (Yac-1 cells) Culture Yac-1 cells in a 25cm ² culture flask, digest with digestive solution (0.25%+0.02% EDTA) after the flask is full, then wash the cells twice with PBS, and re- The cells were suspended in complete medium (modified RPMI 1640+10% FBS), and the cell concentration was adjusted to 2×10 ⁵ /ml.

(3) Cell plating Add 100 μl of mouse spleen lymphocytes at 1×10 ⁷ /ml and 5×10 ⁶ /ml to the 96-well plate respectively, and then add 2×10 ⁵ /ml respectively (effect-to-target ratio 50:1) and 100 μl of Yac-1 cells at 4×10 ⁵ /ml (effect-to-target ratio 25:1), and set effector cell control wells, target cell control wells and blank zero wells, and set three replicates in each well. Place in a 37°C, 5% _CO2 incubator for 5 hours.

(4) Add 50μl of MTT solution (2mg/ml) to each well for color development and incubate for 2-4 hours, centrifuge (1800rpm, 5min) and pour off the culture medium gently, then add 150μl DMSO (containing 4% 1M HCl) , avoid light and shake for 15min.

(5) Detection of NK killing activity The OD value was measured at a wavelength of 490 nm with a microplate reader. The calculation formula of NK cell killing activity is as follows: {1-[(effector cell+target cell well)-effector cell control well]/(target cell control well-blank zero adjustment well)}×100%.

8. Statistical analysis The experimental results are expressed as mean ± standard deviation (Mean ± S.D.), using one-way ANOVA in SPSS (17.0) statistical software, and using Duncan method to carry out multiple comparisons between groups, P <0.05 means the difference is significant, P<0.01 means the difference is extremely significant, Microsoft Office Excel 2007 software for drawing.

2. The results are shown in Figures 6 to 7.

Figure 6 shows that the level of IFN-γ in the vaccine diluent group was significantly higher than that in the control group (P<0.01). IFN-γ is an important cytokine that promotes the body's cellular immune response. Up-regulation of IFN-γ is conducive to the body's cellular immune response and clearance of viral infection.

Figure 7 shows that the activity of NK cells in the vaccine diluent group was significantly higher than that in the control group (P<0.01). NK cells can directly kill invading pathogens and secrete a variety of cytokines to play an immune regulatory role. Increased NK cell activity is conducive to the body's elimination of invading pathogens.

Example 5: Vaccine diluent enhances lymphocyte proliferation and ability to produce cytokines

1. Materials and methods

1. Reagent thimerosal sodium salt (MS), batch number: F20030210, purchased from China Pharmaceutical Group Shanghai Chemical Reagent Company; sodium chloride injection, batch number: B13020601, purchased from Zhejiang Guojing Pharmaceutical Co., Ltd.; porcine pseudorabies virus gB antibody Detection kit, batch number: 09732-FC905, purchased from IDEXX, USA; ginseng stem and leaf saponins (GSLS): batch number: HJ140301, total saponin content 80.23%, containing Rg1 6.0%, Re 16.36%, Rbl 2.4%, Rb2 3.8% , Rf0.1%, Rc 3.70% and Rd 9%, purchased from Jilin Hongjiu Pharmaceutical Co., Ltd. Mouse IFN-γ, IL-10 and IL-12 ELISA kits were purchased from Hangzhou Lianke Biotechnology Co., Ltd.; mouse IL-5 ELISA kits were purchased from eBioscience, USA; RPMI 1640 cell culture medium, batch number: NZF1156 was purchased from Thermo Fisher Biochemical Products (Beijing) Co., Ltd.; premium fetal bovine serum (FBS) (endotoxin <10EU/ml) was purchased from Hyclone Company of the United States; red blood cell lysate, lot number: 20130918, was purchased from Beijing Suo Laibao Biotechnology Co., Ltd.; concanavalin A (ConA), lipopolysaccharide (LPS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), purchased from Sigma, USA -Aldrich Company; Pseudorabies inactivated virus, the wild strain of Pseudorabies was propagated and cultivated by PK cells, the virus content was 106.68TCID50/ml, treated at 60°C for 30min, and stored at -80°C for later use.

2. Preparation of vaccine diluent First prepare 0.002% (weight %) thimerosal normal saline solution as solution A; then prepare 0.012% (weight %) GSLS normal saline solution as solution B. Combine the two liquids A and B in equal weight, and filter the obtained liquid with a 0.22 μm filter membrane to obtain the vaccine dilution.

3. Vaccine Attenuated pseudorabies vaccine, Bartha-K61 strain, batch number: 130901, purchased from Hangzhou Jianliang Veterinary Biological Products Co., Ltd.

4. Animal grouping and immunization 18 ICR mice were randomly divided into 3 groups, 6 in each group. Group 1: dilute vaccine with vaccine diluent, immunize mice by intramuscular injection, the dose of each mouse is 1800TCID ₅₀ ; Group 2: dilute vaccine with normal saline, immunization method and vaccine dosage are the same as group 1; Group 3: use and above-mentioned Both groups injected normal saline to the mice in the same way. Each mouse was injected twice, each injection volume was 0.1 ml, with an interval of 2 weeks.

5. Sampling Spleen lymphocytes of mice were isolated 2 weeks after the second immunization, and lymphocyte proliferation responses and cytokines (IFN-γ, IL-5, IL-10, IL-12) produced by lymphocytes were detected.

6. Lymphocyte proliferation test is carried out according to the following steps:

(1) Separation of the spleen and preparation of mouse spleen lymphocytes.

(2) Red blood cells were removed with erythrocyte lysate, and the cells were washed twice with PBS.

(3) Adjust the cell concentration to 5×10 ⁶ /ml. Add 100 μl of Con A (8 μg/ml), LPS (8 μg/ml) or pseudorabies inactivated virus (106.68TCID50/ml) on a 96-well plate, then add 100 μl of spleen lymphocytes to each well, and set three replicates in each well. Place in a 37°C, 5% _CO2 incubator for 48 hours.

(4) 4 hours before the end of cell culture, 50 μl of MTT solution (2 mg/ml) was added to each well.

(5) Centrifuge the culture plate at 1800 rpm for 5 minutes, discard the supernatant, then add 150 μl of DMSO (containing 4% 1M HCl), and shake for 15 minutes in the dark.

(6) Measure the OD value at a wavelength of 490 nm with a microplate reader. The formula for calculating the lymphocyte stimulation index is as follows: SI=(OD of stimulated well-OD of blank well)/(OD of unstimulated well-OD of blank well).

7. Cytokine detection Use mouse double antibody sandwich ELISA method to detect serum IFN-γ, IL-12, IL-10 and IL-5 levels, the specific steps are as follows:

(1) All reagents taken out of the refrigerator should be brought back to room temperature before use.

(2) Wash the plate twice with 300 μl washing solution per well.

(3) Add 50 μl of detection buffer, 50 μl of sample (or standard or control) and 50 μl of detection antibody to each well in sequence.

(4) Seal the plate with a sealing film, shake at 100 times/min, and incubate at room temperature for 2 hours.

(5) Wash the plate 6 times with 300 μl washing solution.

(6) Add 100 μL horseradish peroxidase-labeled streptavidin to each well.

(7) Seal the plate with a sealing film, shake at 100 times/min, and incubate at room temperature for 45 minutes.

(8) Repeat step (5).

(9) Add 100 μl chromogenic substrate TMB to each well, protect from light, and incubate at room temperature for 10-30 minutes.

(10) Add 100 μl of stop solution to each well and mix well.

(11) Within 30 minutes, measure the OD value with a microplate reader at a wavelength of 450nm, and the reference wavelength is 570nm.

(12) Calculate the content of cytokines with the standard curve.

8. Statistical analysis The experimental results are expressed as mean ± standard deviation (Mean ± S.D.), using one-way ANOVA in SPSS (17.0) statistical software, and using Duncan method to carry out multiple comparisons between groups, P <0.05 means the difference is significant, P<0.01 means the difference is extremely significant, Microsoft Office Excel 2007 software for drawing.

2. The results are shown in Figures 8-9.

It can be seen from Figure 8 that the diluent (GSLS-MS) can significantly enhance the proliferation ability of mouse spleen lymphocytes stimulated by Con A, LPS and PRV antigens, and effectively promote the activation of T and B lymphocytes.

It can be seen from Figure 9 that the diluent (GSLS+MS) can also significantly enhance the production levels of cytokines IFN-γ, IL-12, IL-5 and IL-10 after mouse spleen lymphocytes are stimulated by PRV antigens, indicating that the diluent It can activate balanced Th1 and Th2 immune responses, and has a good adjuvant effect on PRV attenuated vaccine.

Embodiment 6: Vaccine diluent enhances the body's resistance to virulent infection after immunization

1. Materials and methods

1. Reagent thimerosal sodium salt (MS), batch number: F20030210, purchased from China National Pharmaceutical Group Shanghai Chemical Reagent Company; sodium chloride injection, batch number: B13020601, purchased from Zhejiang Guojing Pharmaceutical Co., Ltd.; ginseng stem and leaf saponin (GSLS ): batch number: HJ140301, total saponin content 80.23%, including Rg1 6.0%, Re 16.36%, Rbl 2.4%, Rb2 3.8%, Rf0.1%, Rc 3.70% and Rd 9%, purchased from Jilin Hongjiu Pharmaceutical Co., Ltd. ; The wild strain of porcine pseudorabies was donated by Zhejiang Academy of Agricultural Sciences. After PK cell proliferation and culture, the virus content was 106.68TCID50/ml, and it was stored at -80°C for later use.

2. Preparation of vaccine diluent First prepare 0.002% (weight %) thimerosal normal saline solution as solution A; then prepare 0.012% (weight %) GSLS normal saline solution as solution B. Combine the two liquids A and B in equal weight, and filter the obtained liquid with a 0.22 μm filter membrane to obtain the vaccine dilution.

3. Vaccine Attenuated pseudorabies vaccine, Bartha-K61 strain, batch number: 130901, purchased from Hangzhou Jianliang Veterinary Biological Products Co., Ltd.

4. Animal immunization and challenge Thirty ICR mice were randomly divided into 3 groups, 10 in each group. Group 1: dilute vaccine with vaccine diluent, immunize mice by intramuscular injection, the dose of each mouse is 1800TCID ₅₀ ; Group 2: dilute vaccine with normal saline, immunization method and vaccine dosage are the same as group 1; Group 3: use and above-mentioned Both groups injected normal saline to the mice in the same way. Each mouse was injected twice, each injection volume was 0.1 ml, with an interval of 2 weeks. Two weeks after the second immunization, each mouse was intraperitoneally injected with 5×10 ⁵ TCID ₅₀ wild virus of pseudorabies virus, observed continuously for 10 days and recorded the death of mice in each group.

5. Statistical analysis Experimental result is expressed as mean ± standard deviation (Mean ± S.D.), adopts one-way analysis of variance (one-way ANOVA) in SPSS (17.0) statistical software, and carries out multiple comparison between groups with Duncan method, P <0.05 means that the difference is significant.

2. The results are shown in Figure 10.

It can be seen from Figure 10 that the diluent has a good effect on the challenge of PRV field virus by improving the antibodies, antibody subclasses, IFN-γ, and NK cell killing activity of the mice immunized with the attenuated PRV vaccine, and promoting the activation of T and B lymphocytes. Protective effects. It shows that the diluent is a better adjuvant.

Embodiment 7: vaccine diluent improves the serum antibody level after porcine pseudorabies vaccine immunization

1. Materials and methods

1. Reagent thimerosal sodium salt (MS), batch number: F20030210, purchased from China National Pharmaceutical Group Shanghai Chemical Reagent Company; sodium chloride injection, batch number: B13020601, purchased from Zhejiang Guojing Pharmaceutical Co., Ltd.; ginseng stem and leaf saponin (GSLS ): batch number: HJ140301, total saponin content 80.23%, including Rg1 6.0%, Re 16.36%, Rbl 2.4%, Rb2 3.8%, Rf0.1%, Rc 3.70% and Rd 9%, purchased from Jilin Hongjiu Pharmaceutical Co., Ltd. .

2. Preparation of vaccine diluent First prepare 0.002% (weight %) thimerosal normal saline solution; secondly prepare 0.004%, 0.008% and 0.012% (all weight %) GSLS normal saline solution respectively. Mix thimerosal normal saline solution and GSLS normal saline solution by equal weight, and then filter with a 0.22 μm filter membrane to obtain the following three solutions: 0.001% thimerosal + 0.002% GSLS, 0.001% thimerosal + 0.004% GSLS, 0.001% thimerosal + 0.006 %GSLS, for the three test vaccine dilutions.

3. Vaccine Attenuated pseudorabies vaccine, Bartha-K61 strain, batch number: 130901, purchased from Hangzhou Jianliang Veterinary Biological Products Co., Ltd.

4. Animal grouping and immunization experiments were carried out in a farm in Xiaoshan District, Hangzhou City. Seventy 70-day-old PIC commercial pigs were randomly divided into 5 groups, with 10 pigs in each group. Group 1: Inject 1ml of normal saline; Group 2: According to the instructions of the vaccine, dilute the attenuated pseudorabies vaccine with normal saline, and then inject into the neck muscle, 1ml per head; Group 3-5: Use the three vaccines prepared in step 2 Vaccine diluent was used to dilute the vaccine, and the amount of diluent was in accordance with the amount of normal saline in the vaccine instruction manual. The vaccine dose and immunization method were the same as group 2. Before immunization and 2 and 4 weeks after immunization, 1ml of anterior vena cava blood was collected from each pig, the serum was separated, stored at -80°C, and the serum antibody level was detected by ELISA method.

5. Statistical analysis The experimental results are expressed as mean ± standard deviation (Mean ± S.D.), using one-way ANOVA (one-way ANOVA) in SPSS (17.0) statistical software, and using Duncan method to carry out multiple comparisons between groups, P <0.05 means the difference is significant, P<0.01 means the difference is extremely significant, Microsoft Office Excel 2007 software for drawing.

2. The results are shown in Figure 11.

It can be seen from Figure 11 that the diluent can significantly promote the antibody level induced by the attenuated PRV vaccine, and the antibody level of GSLS 60 μg/ml+MS 0.001% is the highest.

Embodiment 8: Comparative test of ginseng stem and leaf saponins and several preservatives

1. Materials and methods

1. Reagent thimerosal sodium salt (MS), batch number: F20030210, product of China Pharmaceutical Group Shanghai Chemical Reagent Company; sodium chloride injection, batch number: B13020601, product of Zhejiang Guojing Pharmaceutical Co., Ltd.; sodium benzoate (SB), batch number 20101118 , product of Tianjin Bodi Chemical Co., Ltd.; sorbitol (SA), batch number 20101122, product of Tianjin Institute of Photovoltaic Fine Chemicals; ethylparaben (PBB), product of Shanghai Aladdin Reagent Company. Porcine pseudorabies virus gB antibody detection kit, batch number: 09732-FC905, purchased from IDEXX company in the United States; ginseng stem and leaf saponins (GSLS): batch number: HJ140301, total saponin content 80.23%, containing Rg1 6.0%, Re 16.36%, Rbl 2.4%, Rb2 3.8%, Rf 0.1%, Rc 3.70% and Rd 9%, total Rg1, Re, Rd content 31.36%, purchased from Jilin Hongjiu Pharmaceutical Co., Ltd. HRP-labeled goat anti-mouse IgG1 and IgG2a antibodies, lot number: B13020601, were purchased from Santa Cruz Biotechnology Co., Ltd.

2. the preparation of vaccine diluent prepares respectively 0.002% (weight %) thimerosal, sodium benzoate (SB), sorbitol (SA) and ethylparaben (PBB) normal saline solution, is first liquid; Prepare 0.012% again (% by weight) GSLS physiological saline solution is B liquid. Combine A and B in equal weight, or A and normal saline, or B and normal saline, and filter the resulting liquid through a 0.22 μm filter membrane to obtain the following nine solutions: GSLS (0.006%)+MS (0.001%), GSLS(0.006%)+SB(0.001%), GSLS(0.006%)+SA(0.001%), GSLS(0.006%)+PBB(0.001%), MS(0.001%), SB(0.001%), SA( 0.001%), PBB (0.001%) and GSLS (0.006%).

3. Vaccine Attenuated pseudorabies vaccine, Bartha-K61 strain, batch number: 130901, purchased from Hangzhou Jianliang Veterinary Biological Products Co., Ltd.

4. Animal grouping and immunization Sixty ICR mice were randomly divided into 10 groups, 6 in each group. Dilute the vaccine with physiological saline and the above nine solutions respectively to obtain 10 vaccine solutions, each containing 1800 TCID ₅₀ of virus in 0.1 ml. The mice in groups 1 to 10 were immunized by intramuscular injection twice, and each mouse was injected twice, with a volume of 0.1 ml each time, with an interval of 2 weeks.

5. Sampling Blood was collected from the orbit 2 weeks after the second immunization, and the serum was separated to detect antibodies and antibody sublevels.

6. Detection of serum antibodies and their subtypes Use the porcine pseudorabies virus gB antibody detection kit from the American IDEXX company to detect the serum PRV gB antibody level by ELISA antibody blocking method (see Example 1 for specific steps).

7. Statistical analysis Antibody test results are expressed as mean ± standard deviation (Mean ± S.D.). One-way ANOVA in SPSS (l7.0) statistical software was used, and Duncan method was used for multiple comparisons between groups, and Microsoft Office Excel 2007 software was used for drawing.

2. The results are shown in Figure 12.

Figure 12 shows the serum gB antibody levels after diluting the pseudorabies vaccine with different diluents. In each group, the antibody level in the GSLS+MS diluent group was the highest (P<0.05).

Embodiment 9: Containing the comparison of diluents of different doses of thimerosal

1. Materials and methods

1. Reagents Thimerosal sodium salt (MS), lot number: F20030210, product of China Pharmaceutical Group Shanghai Chemical Reagent Company; sodium chloride injection, lot number: B13020601, product of Zhejiang Guojing Pharmaceutical Co., Ltd. Porcine pseudorabies virus gB antibody detection kit, batch number: 09732-FC905, purchased from IDEXX company in the United States; ginseng stem and leaf saponins (GSLS): batch number: HJ140301, total saponin content 80.23%, containing Rg1 6.0%, Re 16.36%, Rbl 2.4%, Rb2 3.8%, Rf 0.1%, Rc 3.70%, and Rd 9%.

2. The preparation of vaccine diluent prepares respectively the thimerosal physiological saline solution of 0.001%, 0.002%, 0.004%, 0.008%, 0.016%, 0.02% (weight %), is A liquid; Saline solution is B liquid. Combine A and B liquids, or B and physiological saline, in equal weights, and filter the resulting liquid through a 0.22 μm filter membrane to obtain the following seven solutions: GSLS (0.006%), GSLS (0.006%)+MS (0.0005%), GSLS(0.006%)+MS(0.001%), GSLS(0.006%)+MS(0.002%), GSLS(0.006%)+MS(0.004%), GSLS(0.006%)+MS(0.008%), GSLS( 0.006%) + MS (0.01%).

3. Vaccine Attenuated pseudorabies vaccine, Bartha-K61 strain, batch number: 130901, purchased from Hangzhou Jianliang Veterinary Biological Products Co., Ltd.

4. Animal grouping and immunization 48 ICR mice were randomly divided into 8 groups, 6 in each group. Dilute the vaccine with physiological saline and the above 7 solutions respectively to obtain 8 vaccine solutions, each containing 1800 TCID ₅₀ of virus in 0.1 ml. The mice in groups 1 to 10 were immunized by intramuscular injection twice, and each mouse was injected twice, with a volume of 0.1 ml each time, with an interval of 2 weeks.

5. Sampling Blood was collected from the orbit 2 weeks after the second immunization, and the serum was separated to detect antibodies and antibody sublevels.

6. Detection of serum antibodies and their subtypes Use the porcine pseudorabies virus gB antibody detection kit from the American IDEXX company to detect the serum PRV gB antibody level by ELISA antibody blocking method (see Example 1 for specific steps).

7. Statistical analysis Antibody test results are expressed as mean ± standard deviation (Mean ± S.D.). One-way ANOVA in SPSS (l7.0) statistical software was used, and Duncan method was used for multiple comparisons between groups, and Microsoft Office Excel 2007 software was used for drawing.

2. The results are shown in Figure 13.

Figure 13 shows the effect of diluent formulations containing different MS on the level of serum gB antibody levels. In each group, the antibody level in the GSLS+MS (0.001%) diluent group was the highest (P<0.05).

Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

Claims (2)

1. the thinner for vaccine containing panaxoside, it is characterized in that being made up of the composition of following weight content：0.006% ginseng stem The thimerosal of leaf saponin or panaxoside, 0.001% or 0.01%, surplus are physiological saline.

2. the thinner for vaccine according to claim 1 containing panaxoside, it is characterized in that：The pH value of the thinner for vaccine is 6~8.