Construction method and application of a k88ac+ attenuated strain of enterotoxigenic Escherichia coli

Abstract

The invention provides a method for constructing a K88ac<+> enterotoxigenic escherichia coli attenuated virus strain. The method includes the following steps that 1, K88ac<+> enterotoxigenic escherichia coli enterotoxin genes are subjected to traceless point mutation, wherein TCT on the LT I gene of K88ac<+> enterotoxigenic escherichia coli without antibiotics resistance genes is mutated into AAA, and correspondingly-encoded No. 63 serine is mutated into lysine (K); 2, the K88ac<+> enterotoxigenic escherichia coli enterotoxin genes are knocked out, wherein on the basis of the first step, the ST II genes in the K88ac<+> enterotoxigenic escherichia coli are knocked out, and therefore the aim of attenuating the K88ac<+> enterotoxigenic escherichia coili is achieved accordingly. The attenuated virus strain constructed with the method is high in adhesivity and long in in-vivo planting time, infection of the pathopoiesia stain can be prevented, and the method can be used for biological preventing of colibacillosis caused by K88ac<+> ETEC.

Description

Technical field

[0001] The present invention relates to a K88ac + The construction method and application of an attenuated strain of enterotoxigenic Escherichia coli belong to the field of bioengineering and animal disease prevention and control. Background technique

[0002] Piglet colibacillosis is also known as piglet yellow and white scour. Data shows that about 50% of piglets die each year due to Enterotoxingenic Escherichia coli (ETEC). Piglet yellow diarrhea generally occurs in piglets within 1 week after delivery, especially within 3-5 days after delivery. Diseased piglets show yellowish white or yellow watery feces. If not treated in time, the mortality rate is as high as 70% or even 100%. The white scour of piglets mostly occurs in 10-30 day old piglets, and the sick pigs are mainly white or off-white loose stools, and the incidence rate can reach about 65%. In addition to the death of piglets, the disease can also affect the growth and development of piglets, reduce f...

Examples

Embodiment 1

[0045] In Example 1, a strain of Escherichia coli isolated from the small intestine of piglets with yellow scour was used to construct K88ac + An attenuated strain of enterotoxigenic Escherichia coli. It includes the following steps:

[0046] 1.K88ac + ETEC separation:

[0047] A strain of Escherichia coli isolated from the small intestine of a 5-day-old piglet with yellow scour of piglets for the first time on a pig farm in Jiangsu using Mackangkai medium. After testing, the E. coli can express K88ac fimbriae, has LT I and ST Ⅱ enterotoxin genes, and has not detected common virulence island genes. It cannot contain ampicillin, tetracycline, gentamicin, kanamycin, and Grow on agar plates containing antibiotics such as cephalosporin, norfloxacin, erythromycin and doxycycline. Named K88ac + ETEC (YZ1205).

[0048] 2. Construction of K88ac+ attenuated strain of enterotoxigenic E. coli:

[0049] (1) LT gene point mutation:

[0050] According to the pUMNK88Ent sequence published by GenBa...

Embodiment 2

[0077] In this example 2, the K88ac constructed in example 1 + The biological characteristics of attenuated strains of enterotoxigenic E. coli were tested as follows:

[0078] 1) Enterotoxin detection

[0079] Prepare improved toxin production medium CAYE medium: tryptone 10g, yeast 3g, sodium chloride 1.25g, dipotassium hydrogen phosphate 5.7g, trace salt mixed solution 0.5mL (5.0% magnesium sulfate, 0.5% manganese chloride, 0.5 % Iron trichloride dissolved in 0.001M sulfuric acid solution) successively dissolved in sterilized distilled water to make the total amount to 500mL. Adjust the pH to 8.5 with 10M sodium hydroxide, autoclave at 121°C for 15min, put it in a refrigerator at 4°C, and add glucose to 0.25% in a sterile method before use.

[0080] K88ac + ETEC (YZ1205), K88ac + ETEC LT(S63K) ΔSTⅡ and DH5α were inoculated in modified CAYE liquid medium, incubated overnight at 37°C, and transferred to 50mL modified CAYE medium at 1:100 the next day, incubated at 37°C for 72 hours,...

Technical field

[0001] The present invention relates to a K88ac + The construction method and application of an attenuated strain of enterotoxigenic Escherichia coli belong to the field of bioengineering and animal disease prevention and control. Background technique

[0002] Piglet colibacillosis is also known as piglet yellow and white scour. Data shows that about 50% of piglets die each year due to Enterotoxingenic Escherichia coli (ETEC). Piglet yellow diarrhea generally occurs in piglets within 1 week after delivery, especially within 3-5 days after delivery. Diseased piglets show yellowish white or yellow watery feces. If not treated in time, the mortality rate is as high as 70% or even 100%. The white scour of piglets mostly occurs in 10-30 day old piglets, and the sick pigs are mainly white or off-white loose stools, and the incidence rate can reach about 65%. In addition to the death of piglets, the disease can also affect the growth and development of piglets, reduce f...

Examples

Embodiment 1

[0045] In Example 1, a strain of Escherichia coli isolated from the small intestine of piglets with yellow scour was used to construct K88ac + An attenuated strain of enterotoxigenic Escherichia coli. It includes the following steps:

[0046] 1.K88ac + ETEC separation:

[0047] A strain of Escherichia coli isolated from the small intestine of a 5-day-old piglet with yellow scour of piglets for the first time on a pig farm in Jiangsu using Mackangkai medium. After testing, the E. coli can express K88ac fimbriae, has LT I and ST Ⅱ enterotoxin genes, and has not detected common virulence island genes. It cannot contain ampicillin, tetracycline, gentamicin, kanamycin, and Grow on agar plates containing antibiotics such as cephalosporin, norfloxacin, erythromycin and doxycycline. Named K88ac + ETEC (YZ1205).

[0048] 2. Construction of K88ac+ attenuated strain of enterotoxigenic E. coli:

[0049] (1) LT gene point mutation:

[0050] According to the pUMNK88Ent sequence published by GenBa...

Embodiment 2

[0077] In this example 2, the K88ac constructed in example 1 + The biological characteristics of attenuated strains of enterotoxigenic E. coli were tested as follows:

[0078] 1) Enterotoxin detection

[0079] Prepare improved toxin production medium CAYE medium: tryptone 10g, yeast 3g, sodium chloride 1.25g, dipotassium hydrogen phosphate 5.7g, trace salt mixed solution 0.5mL (5.0% magnesium sulfate, 0.5% manganese chloride, 0.5 % Iron trichloride dissolved in 0.001M sulfuric acid solution) successively dissolved in sterilized distilled water to make the total amount to 500mL. Adjust the pH to 8.5 with 10M sodium hydroxide, autoclave at 121°C for 15min, put it in a refrigerator at 4°C, and add glucose to 0.25% in a sterile method before use.

[0080] K88ac + ETEC (YZ1205), K88ac + ETEC LT(S63K) ΔSTⅡ and DH5α were inoculated in modified CAYE liquid medium, incubated overnight at 37°C, and transferred to 50mL modified CAYE medium at 1:100 the next day, incubated at 37°C for 72 hours,...

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