CN109248314A

CN109248314A - A kind of preparation method of pig blue-ear disease inactivated vaccine

Info

Publication number: CN109248314A
Application number: CN201811162675.0A
Authority: CN
Inventors: 徐高骁
Original assignee: Nanning Institute
Current assignee: Nanning Institute
Priority date: 2018-09-30
Filing date: 2018-09-30
Publication date: 2019-01-22

Abstract

本发明属于养殖技术领域，尤其是一种猪蓝耳病灭活疫苗的制备方法，包括以下步骤：(1)在MEM基础培养基中加入新生牛血清、中药提取液配制成培养液；(2)使用培养液对Marc‑145细胞进行驯化培养，得到Marc‑145细胞培养液；(3)将猪蓝耳病毒液解入到Marc‑145细胞培养液培养得到病毒液；(4)将病毒液进行澄清、分离、浓缩、纯化；(5)将纯化的病毒液灭活后，与中药提取液配置成灭活疫苗。本发明所使用的中药提取液由茯苓、蒲公英、黄芪、艾草、玉米须、菟丝子、白术、刺五加、淫羊藿制成，能提T细胞、B细胞等细胞因子的活性，促进机体产生抗体。因此，本发明使用中药提取液驯化培养Marc‑145细胞、配置灭活疫苗，能有效提高灭活疫苗对免疫系统的刺激性。The invention belongs to the technical field of breeding, in particular to a preparation method of an inactivated porcine blue-ear disease vaccine, comprising the following steps: (1) adding newborn bovine serum and traditional Chinese medicine extract to a MEM basal medium to prepare a culture solution; (2) The Marc-145 cells are domesticated and cultured using the culture medium to obtain the Marc-145 cell culture fluid; (3) the porcine blue ear virus fluid is decomposed into the Marc-145 cell culture fluid and cultured to obtain the virus fluid; (4) the virus fluid is subjected to Clarify, separate, concentrate and purify; (5) After inactivating the purified virus liquid, configure it with the traditional Chinese medicine extract to form an inactivated vaccine. The traditional Chinese medicine extract used in the present invention is made of Poria cocos, dandelion, astragalus, wormwood, corn silk, dodder, Atractylodes, Acanthopanax senticosus, Epimedium produce antibodies. Therefore, the present invention uses the traditional Chinese medicine extract to domesticate and cultivate Marc-145 cells, and configures the inactivated vaccine, which can effectively improve the stimulation of the inactivated vaccine to the immune system.

Description

A kind of preparation method of pig blue-ear disease inactivated vaccine

Technical field

The invention belongs to cultural technique field, especially a kind of preparation method of pig blue-ear disease inactivated vaccine.

Background technique

Pig blue-ear disease (PRRS), also known as mystery swine disease, new swine disease, pig Epidemic abortion and breathing syndrome, pig reproduction With breathing syndrome, blue otopathy, swine fever epidemic disease etc., it is most important a kind of immunosuppressive disease in China's swine disease, mainly causes to be pregnant Sow Late term abortions, premature labor produce stillborn foetus and the mummification of fetus and increase, there is respiratory symptom in piglet and Preweaning death rate increase with And the immunization machine obstacle of Adult Pig.PRRS virus (PRRSV) belongs to shell type virales, Arteriviridae, is in oval, There is cyst membrane, genome is single strand plus RNA virus；The Principle Target of the virus is Monocyte-macrophages system, at present The primary duplication position for generally believing the virus is the macrophage in schneiderian membrance or upper respiratory tract system, is then followed by blood Ring diffuses to other organs, and is proliferated in mononuclear phagocyte system wherein.

Pig blue-ear disease is a kind of highly contagious disease, and the pig of various kinds, all ages and classes and purposes can infect, but Piglet most easy infection with pregnant sow and within 1 monthly age.Illness pig and with malicious pig be this disease the important infection sources, main propagation Approach is that contact infection, air borne and sperm are propagated, and can also pass through placenta vertical transmission.

Currently, the main means of prevention of pig blue-ear disease is vaccine inoculation, but in practice process, blue-ear disease vaccine is anti- It controls and often will appear immuning failure, the main reason is that it is longer that body generates the neutralizing antibody time, or cannot produce when vaccine immunity The antibody of raw high-titer, and it is possible to infection pig blue-ear disease poison during this period.

Summary of the invention

In order to solve the above technical problems existing in the prior art, the present invention provides a kind of pig blue-ear disease inactivated vaccines Preparation method, particular by following technical scheme realize:

A kind of preparation method of pig blue-ear disease inactivated vaccine, comprising the following steps:

(1) preparation of culture solution: newborn bovine serum is added in MEM basal medium, Chinese medicine extract is configured to cultivate Liquid A and culture solution B；

(2) cell culture: after Marc-145 cell recovery, being linked into culture solution A and cultivated, thin to Marc-145 After born of the same parents grow up to single layer；Continue from selecting pollution-free, morphologically normal Marc-145 cell to be linked into culture solution B in cell monolayer Culture, to get Marc-145 cell culture fluid when cell grows to the 75%~80% of culture solution surface；

(3) Virus culture: pig blue-ear disease venom is linked into Marc-145 cell culture fluid, continue culture to 85% with After there is CPE in upper cell, harvest culture virus liquid；

(4) purify: the virus liquid of harvest is clarified using hollow fiber column, micro-filtration, it is 80-100 times dense after, using dividing Sub- sieve chromatography column purifies filtrate；

(5) inactivate: after the virus liquid using beta-propiolactone inactivation purifying, the Chinese medicine extract mixing for being added 5% or so is matched It is set to inactivated vaccine.

Preferably, the culture solution A is newborn bovine serum and the 15-20% that 10% is added in MEM basal medium Chinese medicine extract.

Preferably, the culture solution B is newborn bovine serum and the 35-45% that 10% is added in MEM basal medium Chinese medicine extract.

Preferably, the Chinese medicine extract is by weight by 10-15 parts of Poria cocos, 6-10 portions of dandelions, 6-10 parts of Huangs Stilbene, 3-5 part wormwood, 10-15 parts of corn stigmas, 3-5 portions of Semen Cuscutaes, 2-4 parts of Rhizoma Atractylodis Macrocephalaes, 2-4 parts of wilsoniis, 2-4 parts of Herba Epimedii.

Preferably, Poria cocos, dandelion, Radix Astragali, Chinese mugwort the preparation of the Chinese medicine extract: are weighed respectively by weight ratio Grass, corn stigma, Semen Cuscutae, Rhizoma Atractylodis Macrocephalae, wilsonii, Herba Epimedii, and crush respectively, then it is mixed and made into medicinal powder；8 are added into medicinal powder After times water impregnates 20-30min, heating decocts 1-1.5h, and filtering obtains filtrate A and the dregs of a decoction；5 times of 80% second is added into the dregs of a decoction Alcohol is heated to 60-70 DEG C, keeps constant temperature 30-40min, and filtering recycles ethyl alcohol, obtains liquor B；Filtrate A and liquor B are mixed Afterwards, the filtering with microporous membrane for the use of aperture being 0.5-10 μm sterilizes to get Chinese medicine extract.

Preferably, the preparation of the pig blue-ear disease venom: 1 monthly age piglet dissect of pig blue-ear disease will be suffered from, acquires lung Dirty and serum is configured to suspension with MEM basal medium after homogenate grinding, adds dual anti-processing, and 1500rmp is centrifuged 20min, takes Supernatant is through 0.20 μm of miillpore filter degerming to get pig blue-ear disease venom.

Preferably, the step (2), cultivation temperature are 36-37 DEG C.

Preferably, the cell density of the Marc-145 cell culture fluid is 6 × 10⁵/ ml~8 × 10⁵/ml

Preferably, the step (3), the access amount of pig indigo plant virus liquid are the 1- of Marc-145 cell culture fluid volume 1.5%.

The beneficial effects of the present invention are: by Poria cocos, dandelion, Radix Astragali, wormwood, corn stigma, Semen Cuscutae, Rhizoma Atractylodis Macrocephalae, thorn five Add, the Chinese medicine extract of Herba Epimedii preparation can promote the generations of the cell factors such as T cell, B cell, monocytes/macrophages, improve The activity of cell factor promotes the generation of antibody；Marc-145 cell is carried out using the culture medium containing Chinese medicine extract tame and docile Change culture, Marc-145 cell can absorb the ingredient in Chinese medicine extract during the growth process, the Marc- after reusing domestication 145 cell culture pig blue-ear disease poison, pig blue-ear disease poison also can promote antibody raw from part traditional Chinese medicine ingredients are wherein absorbed to have The ability of production；Inactivated vaccine is prepared using the pig blue-ear disease venom after Chinese medicine extract and inactivation, further improves inactivation epidemic disease Irritation of the seedling to cell factor.The preparation method of indigo plant otopathy inactivated vaccine provided by the invention, use can promote body to generate The Chinese medicine extract domestication Marc-145 cell of antibody prepares the pig blue-ear disease venom after inactivation, and inactivated vaccine obtained can pierce Swash body and generate antibody, solve inactivated vaccine it is immune when body generate that the neutralizing antibody time is longer, cannot generate high-titer The problem of antibody, has improved inactivated vaccine to the irritation of immune system.

Specific embodiment

It is limited below with reference to specific embodiment technical solution of the present invention is further, but claimed Range is not only limited to made description.

1 Chinese medicine extract of embodiment

Component: 10g Poria cocos, 6g dandelion, 6g Radix Astragali, 3g wormwood, 10g corn stigma, 3g Semen Cuscutae, 2g Rhizoma Atractylodis Macrocephalae, 2g thorn five Add, 2g Herba Epimedii.

Preparation method: Poria cocos, dandelion, Radix Astragali, wormwood, corn stigma, Semen Cuscutae, white is weighed respectively by weight ratio Art, wilsonii, Herba Epimedii, and crush respectively, then it is mixed and made into medicinal powder；After 8 times of water immersion 20min are added into medicinal powder, add Heat decocts 1h, and filtering obtains filtrate A and the dregs of a decoction；5 times of 80% ethyl alcohol is added into the dregs of a decoction, is heated to 60-70 DEG C, keeps constant temperature 30min, filtering recycle ethyl alcohol, obtain liquor B；After filtrate A and liquor B are mixed, filtered using the micropore that aperture is 0.5-10 μm Film filtering, sterilizing obtain Chinese medicine extract 1.

2 Chinese medicine extract of embodiment

Component: 12g Poria cocos, 8g dandelion, 8g Radix Astragali, 4g wormwood, 12g corn stigma, 4g Semen Cuscutae, 3g Rhizoma Atractylodis Macrocephalae, 3g thorn five Add, 3g Herba Epimedii.

Preparation method: Poria cocos, dandelion, Radix Astragali, wormwood, corn stigma, Semen Cuscutae, white is weighed respectively by weight ratio Art, wilsonii, Herba Epimedii, and crush respectively, then it is mixed and made into medicinal powder；After 8 times of water immersion 25min are added into medicinal powder, add Heat decocts 1.2h, and filtering obtains filtrate A and the dregs of a decoction；5 times of 80% ethyl alcohol is added into the dregs of a decoction, is heated to 60-70 DEG C, keeps permanent Warm 35min, filtering recycle ethyl alcohol, obtain liquor B；After filtrate A and liquor B are mixed, the micropore for the use of aperture being 0.5-10 μm Membrane filtration, sterilizing, obtains Chinese medicine extract 2.

3 Chinese medicine extract of embodiment

Component: 15g Poria cocos, 10g dandelion, 10g Radix Astragali, 5g wormwood, 15g corn stigma, 5g Semen Cuscutae, 4g Rhizoma Atractylodis Macrocephalae, 4g thorn Slender acanthopanax, 4g Herba Epimedii.

Preparation method: Poria cocos, dandelion, Radix Astragali, wormwood, corn stigma, Semen Cuscutae, white is weighed respectively by weight ratio Art, wilsonii, Herba Epimedii, and crush respectively, then it is mixed and made into medicinal powder；After 8 times of water immersion 30min are added into medicinal powder, add Heat decocts 1.5h, and filtering obtains filtrate A and the dregs of a decoction；5 times of 80% ethyl alcohol is added into the dregs of a decoction, is heated to 60-70 DEG C, keeps permanent Warm 40min, filtering recycle ethyl alcohol, obtain liquor B；After filtrate A and liquor B are mixed, the micropore for the use of aperture being 0.5-10 μm Membrane filtration, sterilizing, obtains Chinese medicine extract 3.

4 pig blue-ear disease venom of embodiment

1 monthly age piglet dissect of pig blue-ear disease will be suffered from, acquires lungs and serum, cultivated after homogenate grinding with the basis MEM Basigamy is set to suspension, adds dual anti-processing, and 1500rmp is centrifuged 20min, takes supernatant through 0.20 μm of miillpore filter degerming, i.e., Obtain pig blue-ear disease venom.

The preparation of 5 inactivated vaccine of embodiment

Preparation method, comprising the following steps:

(1) preparation of culture solution: 10% newborn bovine serum is added in MEM basal medium, 20% Chinese medicine extract 1 is matched Culture solution A is made, 10% newborn bovine serum is added in MEM basal medium, 45% Chinese medicine extract 1 is configured to culture solution B；

(2) it cell culture: after Marc-145 cell recovery, is linked into culture solution A and is trained in 36-37 DEG C of greenhouse It supports, after Marc-145 cell grows up to single layer；Pollution-free, morphologically normal Marc-145 cell is selected to access from cell monolayer Continue to cultivate in 36-37 DEG C of greenhouse into culture solution B, to get thin when cell grows to the 75%~80% of culture solution surface Born of the same parents' density is 6 × 10⁵/ ml~8 × 10⁵The Marc-145 cell culture fluid of/ml；

(3) Virus culture: pig blue-ear disease venom is linked into Marc-145 cell culture fluid, continue culture to 85% with After there is CPE in upper cell, harvest culture virus liquid；Wherein the access amount of pig indigo plant virus liquid is Marc-145 cell culture fluid volume 1%；

(4) purify: the virus liquid of harvest is clarified using hollow fiber column, micro-filtration, it is 100 times dense after, using molecule Sieve chromatography column purifies filtrate；

6 inactivated vaccine of embodiment

Preparation method, comprising the following steps:

(1) preparation of culture solution: 10% newborn bovine serum is added in MEM basal medium, 18% Chinese medicine extract 2 is matched Culture solution A is made, 10% newborn bovine serum is added in MEM basal medium, 40% Chinese medicine extract 2 is configured to culture solution B；

(2) it cell culture: after Marc-145 cell recovery, is linked into culture solution A and is trained in 36-37 DEG C of greenhouse It supports, after Marc-145 cell grows up to single layer；Pollution-free, morphologically normal Marc-145 cell is selected to access from cell monolayer Continue to cultivate in 36-37 DEG C of greenhouse into culture solution B, when cell grows to the 75%~80% of culture solution surface, obtains cell Density is 6 × 10⁵/ ml~8 × 10⁵The Marc-145 cell culture fluid of/ml；

(3) Virus culture: pig blue-ear disease venom is linked into Marc-145 cell culture fluid, continue culture to 85% with After there is CPE in upper cell, harvest culture virus liquid；Wherein, the access amount of pig indigo plant virus liquid is Marc-145 cell culture liquid Long-pending 1.2%.

(4) purify: the virus liquid of harvest is clarified using hollow fiber column, micro-filtration, it is 90 times dense after, using molecular sieve Chromatographic column purifies filtrate；

7 inactivated vaccine of embodiment

Preparation method, comprising the following steps:

(1) preparation of culture solution: 10% newborn bovine serum is added in MEM basal medium, 15% Chinese medicine extract 3 is matched Culture solution A is made, 10% newborn bovine serum is added in MEM basal medium, 35% Chinese medicine extract 3 is configured to culture solution B；

(2) cell culture: after Marc-145 cell recovery, being linked into culture solution A and cultivated in 36-37 DEG C, After Marc-145 cell grows up to single layer；Pollution-free, morphologically normal Marc-145 cell is selected to be linked into from cell monolayer Continue to cultivate in 36-37 DEG C of greenhouse in culture solution B, when cell grows to the 75%~80% of culture solution surface, obtains cell Density is 6 × 10⁵/ ml~8 × 10⁵The Marc-145 cell culture fluid of/ml；

(3) Virus culture: pig blue-ear disease venom is linked into Marc-145 cell culture fluid, continue culture to 85% with After there is CPE in upper cell, harvest culture virus liquid；Wherein, the access amount of pig indigo plant virus liquid is Marc-145 cell culture liquid Long-pending 1.5%.

(4) purify: the virus liquid of harvest is clarified using hollow fiber column, micro-filtration, it is 80 times dense after, using molecular sieve Chromatographic column purifies filtrate；

1 safety testing of test example

Test method: 20 10 age in days Kunming suckling mouses are bought back, suckling mouse is randomly divided into 4 groups, every group after same raising 10 days 5；Wherein it is inoculated with inactivated vaccine prepared by embodiment 6-7 respectively for 3 groups, another set is inoculated with commercially available pig indigo plant ear inactivated vaccine； Every suckling mouse intramuscular injection 0.1mL vaccine is observed 2 weeks, if second of the suckling mouse intramuscular injection 5mL vaccine of situation without exception every, Continue observation 2 weeks, dissect.

Test result: 4 groups of suckling mouses vaccinate for the first time, are no different paradoxical reaction in 2 weeks；4 groups of suckling mouses inject epidemic disease for the second time Seedling does not occur abnormal response in 2 weeks, does not also occur unusual condition through dissect, illustrate inactivated vaccine prepared by the present invention and city Inactivated vaccine is sold to suckling mouse safety.

2 potency test of test example

Test method: 20 30 age in days piglets are randomly divided into 4 groups, every group 5；1 embodiment 5- is immunized in 1-3 group respectively The inactivated vaccine (5ml/) of 7 preparations, 4 groups are immunized 1 commercially available pig blue-ear disease inactivated vaccine (5ml/)；After immune the 5th, 15, blood sample 30,50,70,90, is acquired respectively, uses the antibody of PRRS virus ELISA antibody assay kit detection serum Level, the results are shown in Table 1:

The PRRSV antibody situation (A of 1 piglet of table_650nm)

5 days

15 days

30 days

50 days

70 days

90 days

1 group

0.98±0.16

1.43±0.38

1.83±0.42

1.23±0.36

0.78±0.27

0.60±0.20

2 groups

0.92±0.13

1.48±0.32

1.96±0.40

1.37±0.57

0.89±0.31

0.64±0.23

3 groups

1.03±0.21

1.52±0.37

1.81±0.38

1.28±0.31

0.91±0.35

0.71±0.18

4 groups

0.98±0.17

1.26±0.31

0.43±0.35

1.71±0.47

1.23±0.28

0.83±0.21

As seen from the table, the piglet of 1-3 group reaches peak value in 30 days or so antibody levels, and 4 groups of piglet was at 50 days or so Antibody level reaches peak value, illustrates that inactivated vaccine prepared by the present invention compared with commercially available pig blue-ear disease inactivated vaccine, can stimulate son The immune response of pig, makes it generate lot of antibodies in a relatively short period of time, can rapidly play a role.

It is important to point out that, above embodiments and test example are only limitted to do further technical solution of the present invention herein Elaboration and understanding, should not be understood as it is further to technical solution of the present invention limited, what those skilled in the art made The innovation and creation of non-protruding essential characteristics and marked improvement still fall within protection category of the invention.

Claims

1. a preparation method of porcine PRRS inactivated vaccine, comprising the following steps:

(1) Preparation of culture solution: add newborn bovine serum and Chinese medicine extract to MEM basal medium to prepare culture solution A and culture solution B;

(2) Cell culture: After the Marc-145 cells are recovered, they are inserted into the medium A for culture, and after the Marc-145 cells grow into a monolayer; the Marc-145 cells with no pollution and normal morphology are selected from the monolayer cells. The cells are inserted into the culture medium B to continue culturing, and when the cells grow to 75% to 80% of the surface of the culture medium, the Marc-145 cell culture medium is obtained;

(3) Virus culture: insert the porcine blue ear virus liquid into the Marc-145 cell culture medium, continue to culture until more than 85% of the cells appear CPE, and then harvest the cultured virus liquid;

(4) Purification: the harvested virus liquid is clarified, microfiltered and concentrated by a hollow fiber column, and after 80-100 times, the filtrate is purified by a molecular sieve chromatography column;

(5) Inactivation: after using β-propiolactone to inactivate the purified virus liquid, add about 5% of the traditional Chinese medicine extract and mix it to prepare an inactivated vaccine.

2. the preparation method of porcine PRRS inactivated vaccine as claimed in claim 1, is characterized in that, described nutrient solution A is to add 10% newborn bovine serum and 15-20% serum in MEM basal medium Chinese herbal extract.

3. the preparation method of porcine PRRS inactivated vaccine as claimed in claim 1, is characterized in that, described nutrient solution B is to add 10% newborn bovine serum and 35-45% serum in MEM basal medium Chinese herbal extract.

4. the preparation method of porcine PRRS inactivated vaccine as claimed in claim 1 is characterized in that, described Chinese medicine extract is composed of 10-15 parts of Poria, 6-10 parts of dandelion, 6-10 parts by weight Astragalus, 3-5 parts of wormwood, 10-15 parts of corn silk, 3-5 parts of dodder, 2-4 parts of Atractylodes, 2-4 parts of Acanthopanax senticosus, 2-4 parts of Epimedium.

5. the preparation method of porcine PRRS vaccine as claimed in claim 4, is characterized in that, the preparation of described Chinese medicine extract: take Poria, dandelion, astragalus, wormwood, corn silk by weight respectively , Dodder, Atractylodes, Acanthopanax senticosus, Epimedium, and pulverized respectively, and then mixed to make medicinal powder; after adding 8 times of water to the medicinal powder, soaked for 20-30min, heated and boiled for 1-1.5h, filtered to obtain filtrate A and medicinal residues; add 5 times of 80% ethanol to the medicinal residues, heat to 60-70°C, keep constant temperature for 30-40min, filter, recover ethanol, and obtain filtrate B; after mixing filtrate A and filtrate B, use a pore size of 0.5- Filter through a 10 μm microporous membrane and sterilize to obtain the Chinese medicine extract.

6. the preparation method of porcine PRRS inactivated vaccine as claimed in claim 1, is characterized in that, the preparation of described porcine blue-ear virus liquid: necropsy of 1-month-old piglets suffering from porcine PRRS, Lungs and serum were collected, homogenized and ground into a suspension with MEM basal medium, treated with double antibodies, centrifuged at 1500 rmp for 20 min, and the supernatant was sterilized by a 0.20 μm microporous filter to obtain a porcine blue ear virus solution. .

7 . The method for preparing an inactivated porcine PRRS vaccine according to claim 1 , wherein in the step (2), the culture temperature is 36-37° C. 8 .

8 . The method for preparing an inactivated porcine PRRS vaccine according to claim 1 , wherein the cell density of the Marc-145 cell culture solution is 6×10 ⁵ /ml～8×10 ⁵ /ml. 9 . .

9. the preparation method of porcine blue ear inactivated vaccine as claimed in claim 1, is characterized in that, described step (3), the access amount of porcine blue virus liquid is 1 % of Marc-145 cell culture liquid volume -1.5%.