阅读说明：本技术 一种通用型猪大肠杆菌病疫苗及其制备方法 (Universal swine colibacillosis vaccine and preparation method thereof ) 是由 袁朗 孙华伟 于 2020-05-15 设计创作，主要内容包括：本发明提供了一种通用型猪大肠杆菌病疫苗,包括基础培养基和添加物,所述基础培养基的组分及质量百分比为：胰蛋白胨1.5%-1.9％、酵母提取物0.2%-0.7％、菌体抗原0.2%-0.7％、水解乳蛋白0.2%-0.7％、粘附素抗原0.1%-0.4％、鞭毛抗原0.2%-0.5％、磷酸二氢钾0.1%-0.7％、磷酸氢二钾0.1%-0.7％、表面抗原0.5%-0.9％、氢氧化铝佐剂0.3%-0.5％、脾氨肽0.2%-0.5%、余量为水,所述添加物为裂解血球全血按体积比为0.8%-1.5％,采用胰蛋白胨、酵母提取物、菌体抗原、水解乳蛋白、粘附素抗原、鞭毛抗原、磷酸二氢钾、磷酸氢二钾、表面抗原和液体佐剂等组成成分,采用多种药物成分以及解血球添加物可以提高疫苗成分的治疗效果和范围,此外添加有脾氨肽可以有效的病猪的免疫力。(The invention provides a universal swine colibacillosis vaccine which comprises a basic culture medium and additives, wherein the basic culture medium comprises the following components in percentage by mass: 1.5 to 1.9 percent of tryptone, 0.2 to 0.7 percent of yeast extract, 0.2 to 0.7 percent of thalli antigen, 0.2 to 0.7 percent of hydrolyzed milk protein, 0.1 to 0.4 percent of adhesin antigen, 0.2 to 0.5 percent of flagellum antigen, 0.1 to 0.7 percent of potassium dihydrogen phosphate, 0.1 to 0.7 percent of dipotassium hydrogen phosphate, 0.5 to 0.9 percent of surface antigen, 0.3 to 0.5 percent of aluminum hydroxide adjuvant, 0.2 to 0.5 percent of spleen amino peptide and the balance of water, wherein the additives are components of lysed whole blood cells according to the volume ratio of 0.8 to 1.5 percent, the tryptone, the yeast extract, the thalli antigen, the hydrolyzed milk protein, the adhesin antigen, the flagellum antigen, the potassium dihydrogen phosphate, the dipotassium hydrogen phosphate, the surface antigen, liquid adjuvant and the like are adopted, and the treatment effect and the range of the components can be improved by adopting various pharmaceutical components and the additives of the lysed blood, in addition, spleen aminopeptide can be added to effectively improve the immunity of sick pigs.)

1. A general type swine colibacillosis vaccine is characterized in that: the culture medium comprises a basic culture medium and additives, wherein the basic culture medium comprises the following components in percentage by mass: 1.5-1.9% of tryptone, 0.2-0.7% of yeast extract, 0.2-0.7% of thalline antigen, 0.2-0.7% of hydrolyzed milk protein, 0.1-0.4% of adhesin antigen, 0.2-0.5% of flagellum antigen, 0.1-0.7% of potassium dihydrogen phosphate, 0.1-0.7% of dipotassium hydrogen phosphate, 0.5-0.9% of surface antigen, 0.3-0.5% of aluminum hydroxide adjuvant, 0.2-0.5% of spleen amino peptide and the balance of water, wherein the additive is 0.8-1.5% of lysed whole blood cells by volume ratio.

2. The universal swine colibacillosis vaccine of claim 1, wherein: the culture medium comprises a basic culture medium and additives, wherein the basic culture medium comprises the following components in percentage by mass: tryptone 1.7%, yeast extract 0.5%, thallus antigen 0.5%, hydrolyzed milk protein 0.5%, adhesin antigen 0.3%, flagellum antigen 0.3%, potassium dihydrogen phosphate 0.5%, dipotassium hydrogen phosphate 0.4%, surface antigen 0.7%, liquid adjuvant 0.4%, spleen amino peptide 0.3%, and balance of water, wherein the additive is 1.1% of lysed whole blood according to volume ratio.

3. The universal swine colibacillosis vaccine of claim 2, wherein: the adhesin antigen employs one or more of K88, K99, 987P, and F41.

4. The universal swine colibacillosis vaccine of claim 2, wherein: the liquid adjuvant adopts a water adjuvant which is aluminum hydroxide and a MONTANIDE & lt & gtGEL adjuvant.

5. The method for preparing the general swine colibacillosis vaccine according to claims 1-4, wherein the method comprises the following steps: the preparation method comprises the following steps:

step 1: extracting pathogenic bacteria, namely extracting corresponding bacteria from the large intestine of a sick pig of each farm respectively aiming at the farms with demands, marking and reserving the bacteria for use, then culturing, inactivating, centrifuging and resuspending the bacteria of each farm to obtain escherichia coli thalli, and performing targeted low-yield solid culture;

step 2: and (3) culturing the escherichia coli: respectively scribing bacteria of each farm on a nutrition plate, transferring the bacteria to an identification culture medium after the bacteria grow out, or directly scribing the bacteria on the identification culture medium, putting the bacteria into a 37 ℃ thermostat, picking a plurality of related bacterial colonies and putting the bacterial colonies into nutrient broth for amplification culture after the bacteria which accord with the regulation grow out, respectively coating and inoculating escherichia coli liquid of different farms which grow well onto the nutrition plate after 18-30 hours, and putting a certain amount of coated escherichia coli plates into a 37 ℃ thermostat for culture for 18-30 hours according to the number of poultry of each farm;

and step 3: deep treatment of escherichia coli liquid and vaccine preparation: taking out the well-grown escherichia coli plate, soaking escherichia coli on the plate in normal saline in an ultra-clean bench, lightly scraping lawn by using a coater, washing and collecting to obtain escherichia coli liquid of different farms, sequentially putting a mixture of tryptone, yeast extract, thalli antigen, lactoprotein hydrolysate, adhesin antigen, flagellar antigen, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, surface antigen, liquid adjuvant, splenic amino peptide and lysed blood cells into the escherichia coli liquid according to the proportion, putting the mixture into a shaking table at 37 ℃ for oscillation inactivation for 3-5 days, and performing inactivation detection, wherein the centrifugal stirring speed is 4000-;

and 4, step 4: and (3) vaccine effect detection: selecting inactivated bacteria liquid to carry out nutrition plate streak detection, observing whether escherichia coli grows or not after 24-48 hours, wherein no bacterial colony is generated on the nutrition plate after the inactivation is qualified, and the escherichia coli vaccine is divided into escherichia coli water vaccine or oil emulsion inactivated vaccine, and the escherichia coli water vaccine directly dilutes escherichia coli thallus through normal saline to prepare the vaccine.

And 5: and (3) bottling the vaccine: and bottling the medicinal solution through a production line according to the proportion, carrying out vacuum sealing, and finally drying and storing the bottled medicament at low temperature.

6. The universal swine colibacillosis vaccine of claim 5, wherein: the additive adopts a jugular vein blood collection or carotid artery blood discharge method to aseptically adopt robust pig blood in a non-affected area and aseptically defibrinate the lysed corpuscles to be added into a basic culture medium.

Technical Field

The invention relates to the technical field of animal vaccines, in particular to a universal swine colibacillosis vaccine and a preparation method thereof.

Background

Porcine colibacillosis is a piglet intestinal infectious disease caused by pathogenic escherichia coli. Three diseases, namely yellow scour of piglets, white scour of piglets and edema disease, are common, and the enteritis and the enterotoxemia are characterized. The yellow scour of piglets is also called as early-onset colibacillosis, and is an acute and highly lethal intestinal infectious disease of piglets aged 1-7 days. The white scour of piglets is caused by escherichia coli, and is an acute digestive tract infectious disease frequently occurring in piglets of about 10-30 days old. Clinically, the stink-discharging grey-white and thin atheromatous excrement is mainly characterized by high morbidity and low mortality, is a common disease of pigs with more complications, and needs to be prevented by a pig colibacillosis vaccine.

The existing swine colibacillosis vaccine has the following problems in the process of preventing and treating the swine: (1) the existing vaccine for the swine colibacillosis has single component composition, has strong pertinence to a prevention object, cannot meet the prevention and treatment of pigs in different stages, and has poor universality; (2) the existing culture and preparation methods for the swine colibacillosis vaccine are relatively fixed, and the drug effect of the vaccine is influenced.

Disclosure of Invention

Aiming at the defects, the invention aims to provide the vaccine for the general swine colibacillosis and the preparation method thereof, and the vaccine has high drug effect and enlarges the application range of the vaccine by adding a plurality of drug combination components and improving the cultivation process.

The invention is realized by the following technical scheme:

a general type swine colibacillosis vaccine comprises a basic culture medium and additives, wherein the basic culture medium comprises the following components in percentage by mass: 1.5-1.9% of tryptone, 0.2-0.7% of yeast extract, 0.2-0.7% of thalline antigen, 0.2-0.7% of hydrolyzed milk protein, 0.1-0.4% of adhesin antigen, 0.2-0.5% of flagellum antigen, 0.1-0.7% of potassium dihydrogen phosphate, 0.1-0.7% of dipotassium hydrogen phosphate, 0.5-0.9% of surface antigen, 0.3-0.5% of aluminum hydroxide adjuvant, 0.2-0.5% of spleen amino peptide and the balance of water, wherein the additive is 0.8-1.5% of lysed whole blood cells by volume ratio.

The improvement of the technical scheme comprises a basic culture medium and additives, wherein the basic culture medium comprises the following components in percentage by mass: tryptone 1.7%, yeast extract 0.5%, thallus antigen 0.5%, hydrolyzed milk protein 0.5%, adhesin antigen 0.3%, flagellum antigen 0.3%, potassium dihydrogen phosphate 0.5%, dipotassium hydrogen phosphate 0.4%, surface antigen 0.7%, liquid adjuvant 0.4%, spleen amino peptide 0.3%, and balance of water, wherein the additive is 1.1% of lysed whole blood according to volume ratio.

As an improvement of the technical scheme, the adhesin antigen adopts one or more of K88, K99, 987P and F41.

As an improvement of the technical scheme, the liquid adjuvant adopts a water adjuvant, and the water adjuvant is aluminum hydroxide and a MONTANIDE ™ Gel adjuvant.

The preparation method comprises the following steps:

step 1: extracting pathogenic bacteria, namely extracting corresponding bacteria from the large intestine of a sick pig of each farm respectively aiming at the farms with demands, marking and reserving the bacteria for use, then culturing, inactivating, centrifuging and resuspending the bacteria of each farm to obtain escherichia coli thalli, and performing targeted low-yield solid culture;

step 2: and (3) culturing the escherichia coli: respectively scribing bacteria of each farm on a nutrition plate, transferring the bacteria to an identification culture medium after the bacteria grow out, or directly scribing the bacteria on the identification culture medium, putting the bacteria into a 37 ℃ thermostat, picking a plurality of related bacterial colonies and putting the bacterial colonies into nutrient broth for amplification culture after the bacteria which accord with the regulation grow out, respectively coating and inoculating escherichia coli liquid of different farms which grow well onto the nutrition plate after 18-30 hours, and putting a certain amount of coated escherichia coli plates into a 37 ℃ thermostat for culture for 18-30 hours according to the number of poultry of each farm;

and step 3: deep treatment of escherichia coli liquid and vaccine preparation: taking out the well-grown escherichia coli plate, soaking escherichia coli on the plate in normal saline in an ultra-clean bench, lightly scraping lawn by using a coater, washing and collecting to obtain escherichia coli liquid of different farms, sequentially putting a mixture of tryptone, yeast extract, thalli antigen, lactoprotein hydrolysate, adhesin antigen, flagellar antigen, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, surface antigen, liquid adjuvant, splenic amino peptide and lysed blood cells into the escherichia coli liquid according to the proportion, putting the mixture into a shaking table at 37 ℃ for oscillation inactivation for 3-5 days, and performing inactivation detection, wherein the centrifugal stirring speed is 4000-;

and 4, step 4: and (3) vaccine effect detection: selecting inactivated bacteria liquid to carry out nutrition plate streak detection, observing whether escherichia coli grows or not after 24-48 hours, wherein no bacterial colony is generated on the nutrition plate after the inactivation is qualified, and the escherichia coli vaccine is divided into escherichia coli water vaccine or oil emulsion inactivated vaccine, and the escherichia coli water vaccine directly dilutes escherichia coli thallus through normal saline to prepare the vaccine.

And 5: and (3) bottling the vaccine: and bottling the medicinal solution through a production line according to the proportion, carrying out vacuum sealing, and finally drying and storing the bottled medicament at low temperature.

As an improvement of the technical scheme, the additive adopts a jugular vein blood collection or carotid artery blood discharge method to aseptically collect robust pig blood in a non-affected area and aseptically defibrinate the lysed corpuscles to be added into a basic culture medium.

The invention has the beneficial effects that:

(1) the invention adopts components such as tryptone, yeast extract, thalli antigen, hydrolyzed milk protein, adhesin antigen, flagellar antigen, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, surface antigen, liquid adjuvant and the like, adopts various medicinal components and hemolyzing additives to improve the treatment effect and range of vaccine components, and can effectively improve the immunity of sick pigs by adding the spleen aminopeptide.

(2) The scheme adopts a multi-step differentiated culture mode for the culture and preparation modes of the vaccine, so that the treatment effect of the vaccine on pigs in different stages can be improved, and the vaccine finished product has better universality.

Detailed Description