CN106987549B - Culture medium and corresponding production method of broad-spectrum bacterin for preventing poultry colibacillosis - Google Patents
Culture medium and corresponding production method of broad-spectrum bacterin for preventing poultry colibacillosis Download PDFInfo
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- 239000001963 growth medium Substances 0.000 title claims abstract description 57
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 29
- 244000144977 poultry Species 0.000 title claims abstract description 18
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 37
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 37
- 230000014509 gene expression Effects 0.000 claims abstract description 29
- 239000000843 powder Substances 0.000 claims abstract description 11
- 150000001413 amino acids Chemical class 0.000 claims abstract description 8
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- 235000003891 ferrous sulphate Nutrition 0.000 claims abstract description 7
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims abstract description 7
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims abstract description 7
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims abstract description 7
- 229960001763 zinc sulfate Drugs 0.000 claims abstract description 7
- 229910000368 zinc sulfate Inorganic materials 0.000 claims abstract description 7
- 238000000855 fermentation Methods 0.000 claims description 17
- 230000003321 amplification Effects 0.000 claims description 13
- 230000001580 bacterial effect Effects 0.000 claims description 13
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 13
- 241000894006 Bacteria Species 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 12
- 230000004151 fermentation Effects 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 12
- 241000271566 Aves Species 0.000 claims description 10
- 241000588724 Escherichia coli Species 0.000 claims description 7
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 230000001939 inductive effect Effects 0.000 claims description 6
- 239000008101 lactose Substances 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 230000002265 prevention Effects 0.000 claims description 5
- 238000009423 ventilation Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- 238000010907 mechanical stirring Methods 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- 238000005273 aeration Methods 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 2
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 2
- 239000008103 glucose Substances 0.000 claims description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 2
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 2
- 238000013341 scale-up Methods 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 11
- 238000009776 industrial production Methods 0.000 abstract description 5
- 239000002994 raw material Substances 0.000 abstract description 4
- 239000000463 material Substances 0.000 abstract description 3
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- 239000002609 medium Substances 0.000 description 12
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- 210000003746 feather Anatomy 0.000 description 5
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- 230000001954 sterilising effect Effects 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 206010006811 Bursitis Diseases 0.000 description 1
- 206010007882 Cellulitis Diseases 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 206010022678 Intestinal infections Diseases 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 206010071301 Perihepatitis Diseases 0.000 description 1
- 241000286209 Phasianidae Species 0.000 description 1
- 208000007893 Salpingitis Diseases 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
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- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
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- 206010034674 peritonitis Diseases 0.000 description 1
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- 230000009885 systemic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
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Abstract
The invention discloses a culture medium, which can promote the growth of broad-spectrum bacterins after ferrous sulfate, zinc sulfate and amino acid powder are added, the viable count and the protein expression of the bacterins are high, the material compatibility is reasonable, and the raw material price is low; the invention also provides a production method of the broad-spectrum bacterin for preventing the poultry colibacillosis by using the culture medium, and the method has the advantages of simple process, easy control and suitability for industrial production. The invention is suitable for producing broad-spectrum bacterins for preventing poultry colibacillosis.
Description
Technical Field
The invention belongs to the field of veterinary bacterins, and relates to a culture medium and a production method of broad-spectrum bacterins for preventing poultry colibacillosis by applying the culture medium.
Background
Avian Colibacillosis (AC) is a general term for local or systemic extra-intestinal infections of chickens, turkeys and other birds caused by Avian Pathogenic Escherichia Coli (APEC), and is clinically common in cases of colibacillosis, cellulitis, salpingitis, peritonitis, swollen head syndrome, septicemia (pericarditis, perihepatitis, bursitis), and the like. The existing formula of the broad-spectrum vaccine preparation method for preventing poultry colibacillosis is limited to conventional fermentation culture media such as LB culture medium, yeast powder, peptone, inorganic salt and the like which are commonly used in a laboratory shake flask, the viable count of a single batch of fermentation vaccine only reaches 4.0 multiplied by 109CFU/mL, the protein expression quantity is less than or equal to 0.12mg/mL, the preparation quantity is small, only 50 feathers are used per milliliter, and the requirements of amplification large-scale quantitative production and market cannot be met. Therefore, the development of a production method which has stable performance, can improve the number of live bacteria of the vaccine and has high protein expression is urgently needed.
At present, corresponding researches on construction of genetic engineering bacteria used by poultry escherichia coli vaccines, gene expression and the like are carried out at home and abroad, but the finally expressed viable bacteria number and protein expression quantity are relatively low, and the effect is poor.
Disclosure of Invention
The invention aims to provide a culture medium, the material compatibility in the culture medium is reasonable, the raw material price is low, and the culture medium is applied to the broad-spectrum vaccine production for preventing poultry colibacillosis and can obtain higher vaccine viable count and vaccine protein expression quantity;
the invention also aims to provide a method for producing broad-spectrum bacterins for preventing poultry colibacillosis by using the culture medium, which is simple, easy to control and suitable for industrial production.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the culture medium is characterized by comprising the following components in parts by weight:
0.5-1.5 parts of dipotassium hydrogen phosphate, 0.1-0.5 part of monopotassium phosphate, 0.2-0.5 part of ammonium sulfate, 0.0005-0.001 part of calcium chloride, 0.1-0.6 part of magnesium sulfate, 1-2.5 parts of peptone, 0.5-2 parts of yeast powder, 1-2.5 parts of glucose, 0.2-0.5 part of sodium chloride, 0.001-0.005 part of ferrous sulfate, 0.005-0.01 part of zinc sulfate and 100 parts of water.
As the limitation of the culture medium, the culture medium also comprises 1-2 parts by weight of amino acid powder.
The invention also provides a production method of the broad-spectrum bacterin for preventing poultry colibacillosis, which is sequentially carried out according to the following steps:
(1) preparing an escherichia coli strain containing the recombinant plasmid, and freezing and storing;
(2) the culture medium provided by the invention is adopted to carry out expanded culture on strains;
(3) APECtsh protein was expressed induced using a mechanically stirred aerated fermentor.
As a limitation of the production method of the present invention:
the amplification culture is divided into three stages, and the specific steps are as follows:
(1) first-stage amplification culture: adding the bacterial liquid into a culture medium, and performing shake culture at 220rpm and 37 ℃ for 4-8h;
(2) second-stage amplification culture: adding the bacterial liquid treated in the step (1) into a culture medium, and performing shake culture at 220rpm and 37 ℃ for 4-8h;
(3) third-stage amplification culture: adding the bacterial liquid treated in the step (2) into a culture medium, culturing in a fermentation tank, and maintaining the stirring speed of 500-700rpm and the maximum aeration ratio of 1vvm in the culture process, and culturing at 37 ℃ for 5-10 h; continuing to culture at 26 deg.C for 8h, cooling to 26 deg.C, and adding lactose with final concentration of 10 g/L;
the three-stage amplification culture of the step (3) comprises the following specific steps:
a mechanical stirring ventilation fermentation tank is used, the stirring speed is 500-; culturing at 26 deg.C for 8 hr, cooling to 26 deg.C, and adding lactose with final concentration of 10 g/L.
The production method of the broad-spectrum vaccine for preventing poultry colibacillosis has a limitation, and the escherichia coli refers to BL21 engineering bacteria containing a recombinant plasmid pEASY-E1.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the technical progress that:
the culture medium provided by the invention has reasonable material compatibility and low raw material price, can obtain higher live bacterial count and vaccine protein expression quantity when being applied to broad-spectrum vaccine production for preventing poultry colibacillosis, and the obtained live bacterial count is more than or equal to 1.0 × 1010CFU/mL; the expression level of the obtained bacterin protein is more than or equal to 0.3mg/mL, and each milliliter of bacterin protein can be used by 200 feathers;
the production method of broad-spectrum bacterins for preventing poultry colibacillosis by applying the culture medium is simple in process, easy to control and suitable for industrial production.
The invention is suitable for producing broad-spectrum bacterins for preventing poultry colibacillosis.
The present invention will be described in further detail with reference to specific examples.
Drawings
FIG. 1 is a flow chart of a method for producing broad-spectrum bacterins for the prevention of colibacillosis in poultry;
FIG. 2 is an SDS-PAGE electrophoresis chart of APECtsh protein expression;
in the figure: M-Mark, 1-fermentation broth with APECtsh protein expression using # 1 medium, 2-fermentation broth with APECtsh protein expression using # 2 medium, 3-fermentation broth with APECtsh protein expression using # 3 medium, 4-fermentation broth with APECtsh protein expression using # 4 medium, 5-fermentation broth with APECtsh protein expression using # 5 medium, 6-negative control (fermentation broth without APECtsh protein expression using conventional LB medium).
Detailed Description
The reagents and methods used in the following examples are conventional reagents and conventional methods unless otherwise specified.
EXAMPLES 1-5 culture Medium
Examples 1 to 5 are each a liquid medium prepared from the following raw materials as shown in the following table:
TABLE 1 culture Medium compounding Table
EXAMPLE 6 production of broad-spectrum vaccine for prevention of avian colibacillosis
This example is a method for producing broad-spectrum bacterins for preventing avian colibacillosis, the flow chart of the whole production is shown in FIG. 1, and the method is sequentially carried out according to the following steps:
(61) preparing an escherichia coli strain containing the recombinant plasmid (BL 21 engineering bacteria containing the recombinant plasmid pEASY-E1), and freezing and storing;
the frozen preserved strain was treated as follows:
(6a) thawing the strain at 4 ℃, culturing overnight in an LB culture medium added with 100 mug/mL AMP at 220rpm and 37 ℃ to recover the strain, and then coating the strain on an LB solid culture medium to culture a single colony;
(6b) picking a single colony to be cultured in 2mL LB culture medium at 220rpm and 37 ℃ for 7 h; then transferring the mixture to 100mL LB culture medium, and culturing for 7h at 37 ℃ and 220 rpm;
wherein:
LB liquid culture medium configuration: weighing various reagents, putting the reagents into a 250ml triangular flask, adding 100ml of purified water to dissolve the reagents, and sterilizing the reagents in an autoclave at 121 ℃ for 20min to obtain the product.
The LB solid medium is used for streaking plate culture monoclonal colonies after strain recovery, and is configured as follows:
1g of sodium chloride, 1g of peptone, 0.5g of yeast, 0.8 g of agar and 100 g of water;
weighing the reagents, placing into a 250ml triangular flask, adding 100ml purified water, dissolving, and sterilizing at 121 deg.C for 20min in an autoclave;
(62) the culture medium provided in example 1 was used to perform scale-up culture of the strain;
the amplification culture is a three-stage culture, which comprises the following specific steps:
(6A) first-stage amplification culture: adding the bacterial liquid into a culture medium, and performing shake culture at 220rpm and 37 ℃ for 6h to obtain a bacterial liquid R;
(6B) second-stage amplification culture: adding the bacterial liquid R into a culture medium, and performing shake culture at 220rpm and 37 ℃ for 7h to obtain a bacterial liquid S;
(6C) third-stage amplification culture: adding the bacterial liquid S into a culture medium, culturing in a fermentation tank, keeping the stirring speed at 500rpm and the maximum aeration ratio at 1vvm during the culture process, and culturing at 37 ℃ for 5 h; continuing to culture at 26 deg.C for 8h, cooling to 26 deg.C, and adding lactose with final concentration of 10 g/L;
(63) inducing and expressing APECtsh protein by using a mechanical stirring ventilation fermentation tank;
culturing for 10h at 37 ℃ by using a mechanical stirring ventilation fermentation tank with the stirring speed of 500rpm and the maximum ventilation ratio of 1 vvm; culturing at 26 deg.C for 8 hr, cooling to 26 deg.C, and adding lactose with final concentration of 10 g/L.
The production method of the broad-spectrum bacterin for preventing the poultry colibacillosis provided by the embodiment has the advantages of simple process, easiness in control and suitability for industrial production.
Examples 7-10 production of broad-spectrum bacterins for prevention of colibacillosis in avian species
Examples 7-10 each provide a method for producing broad-spectrum vaccines for the prevention of colibacillosis in birds, which are similar to example 6, except that:
firstly, the related technical parameters are different in the production process;
the media used in examples 7 to 10 were the media provided in examples 2, 3, 4 and 5, respectively. Wherein, the specific values of the technical parameters are shown in the following table:
TABLE 2 production Process parameters Table
The production method of broad-spectrum bacterins for preventing poultry colibacillosis provided in examples 7-10 is simple in process, easy to control and suitable for industrial production.
EXAMPLE 11 results test
The products obtained in examples 6-10 were tested to obtain a vaccine with a viable count of 1.0 × 10 or more10CFU/mL; the expression level of the obtained bacterin protein is more than or equal to 0.3mg/mL, and each milliliter of bacterin protein can be used for 200 feathers.
Example 12 comparative experiment
To verify the effect of the media provided by the present invention, this example provides 5 media, wherein: the culture medium # 1 is the culture medium provided in example 1, and the culture medium # 2-5 is similar to the culture medium # 1 in the following ingredients, except that:
the 2# culture medium does not contain amino acid powder, ferrous sulfate and zinc sulfate;
the No. 3 culture medium does not contain amino acid powder and zinc sulfate;
no. 4 medium does not contain amino acid powder;
no. 5 medium contained no ferrous sulfate;
the 6# medium used a conventional LB medium as a negative control.
The above culture medium is prepared, and then sterilized by high pressure steam for 15 min.
Broad-spectrum bacterins were produced according to the method provided in example 6, and the number of live bacteria and the protein expression of bacterins cultured in various media were counted when the bacterins were removed from the jar, and the results were as follows:
the number of live bacteria of the vaccine obtained from the No. 1 culture medium is 1.3 × 1010CFU/mL, the protein expression level is 0.5mg/mL, and each milliliter can be used for 250 feathers;
the number of viable bacteria of the vaccine obtained from the 2# culture medium is 4.2 × 109CFU/mL, protein expression of 0.08mg/mL, protein expression SDS-PAGE compare see figure 2, each mL only for 50 feather use, the production level is far lower than the culture medium production level that the invention provides;
the number of viable bacteria of the vaccine obtained from the 3# culture medium is 3.5 × 109CFU/mL, protein expression amount of 0.1mg/mL, protein expression SDS-PAGE contrast see figure 2;
the number of viable bacteria of the vaccine obtained from the No. 4 culture medium is 3.2 × 109CFU/mL, protein expression amount of 0.12mg/mL, protein expression SDS-PAGE contrast see figure 2;
the number of viable bacteria of the vaccine obtained from the 5# control group culture medium is 3.2 × 109CFU/mL, protein expression amount of 0.11mg/mL, protein expression SDS-PAGE contrast see figure 2;
in summary, only one or two of ferrous sulfate, zinc sulfate and amino acid powder are added to the culture medium of control group # 3, # 4 and # 5, and the viable count and protein expression of the vaccine obtained by fermentation culture are significantly lower than those of the culture medium (provided by the invention in example 1) with all three components added.
Example 13 comparative experiment
In this example, the same procedure as in example 12 was adopted, and the culture medium # 1 in example 12 was replaced with the culture medium provided in examples 2 to 5, respectively, and accordingly, the culture medium # 2 to 5 was adjusted in the same manner. The comparative results of this example demonstrate that: the culture medium of the 3#, 4#, and 5# control group is only added with one or two of ferrous sulfate, zinc sulfate, and amino acid powder, and the viable count and protein expression of the vaccine obtained by fermentation culture are significantly lower than those of the culture medium (provided by the culture medium provided by embodiments 2, 3, 4, and 5 of the invention) with all three components.
The embodiments 1-10 are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, so that any person skilled in the art can make modifications or changes to the equivalent embodiments by using the above technical teaching. However, simple modifications, equivalent changes and modifications of the above embodiments may be made without departing from the technical spirit of the claims of the present invention, and the scope of the claims of the present invention may be protected.
Claims (5)
1. A culture medium for inducing and expressing APECtsh protein is characterized by comprising the following components in parts by weight:
0.5-1.5 parts of dipotassium hydrogen phosphate, 0.1-0.5 part of monopotassium phosphate, 0.2-0.5 part of ammonium sulfate, 0.0005-0.001 part of calcium chloride, 0.1-0.6 part of magnesium sulfate, 1-2.5 parts of peptone, 0.5-2 parts of yeast powder, 1-2.5 parts of glucose, 0.2-0.5 part of sodium chloride, 0.001-0.005 part of ferrous sulfate, 0.005-0.01 part of zinc sulfate, 100 parts of water and 1-2 parts of amino acid powder.
2. The production method of broad-spectrum bacterin for preventing poultry colibacillosis is characterized by sequentially carrying out the following steps:
(1) preparing an escherichia coli strain containing the recombinant plasmid, and freezing and storing;
(2) using the culture medium for inducing and expressing APECtsh protein as claimed in claim 1, carrying out scale-up culture on strains;
(3) APECtsh protein was expressed induced using a mechanically stirred aerated fermentor.
3. The method for producing broad-spectrum bacterins for preventing avian colibacillosis according to claim 2, wherein the expanded culture is divided into three stages, and the specific steps are as follows:
(1) first-stage amplification culture: adding the bacterial liquid into the culture medium for inducing expression of APECtsh protein according to claim 1, and performing shaking culture at 220rpm and 37 ℃ for 4-8h;
(2) second-stage amplification culture: adding the bacterial liquid treated in the step (1) into the culture medium for inducing and expressing APECtsh protein, which is disclosed in the claim 1, and carrying out shake culture at 220rpm and 37 ℃ for 4-8h;
(3) third-stage amplification culture: adding the bacterial liquid treated in the step (2) into the culture medium for inducing and expressing APECtsh protein, which is described in the claim 1, and culturing in a fermentation tank, wherein the stirring speed is 500-700rpm, the maximum aeration ratio is 1vvm, and the culture is carried out for 5-10h at 37 ℃; culturing at 26 deg.C for 8 hr, cooling to 26 deg.C, and adding lactose with final concentration of 10 g/L.
4. The method for producing broad-spectrum bacterin for preventing avian colibacillosis according to claim 2, wherein the step (3) comprises the following steps:
a mechanical stirring ventilation fermentation tank is used, the stirring speed is 500-; culturing at 26 deg.C for 8 hr, cooling to 26 deg.C, and adding lactose with final concentration of 10 g/L.
5. The method for producing broad-spectrum bacterins for the prevention of colibacillosis in avian according to any one of claims 2 to 4, wherein: the Escherichia coli refers to BL21 engineering bacteria containing a recombinant plasmid pEASY-E1.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1822855A (en) * | 2003-05-14 | 2006-08-23 | 惠氏公司 | Avian e. coli vaccine for protection against colibacillosis |
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