Method for constructing K88ac<+> enterotoxigenic escherichia coli attenuated virus strain and application

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 kind of K88ac + The invention relates to a construction method and application of an attenuated strain of enterotoxigenic Escherichia coli, belonging to the fields of bioengineering and animal disease prevention and control. Background technique [0002] Piglet colibacillosis is also known as piglet yellow and white diarrhea. According to data, about 50% of piglets die each year due to enterotoxigenic Escherichia coli (ETEC). Piglet yellow scour generally occurs in piglets within 1 week after delivery, especially within 3-5 days after delivery. The diseased piglets manifest as yellowish white or yellow watery feces. If not treated in time, the mortality rate is as high as 70% or even 100%. Piglet pullorum occurs mostly in 10-30-day-old piglets, and the diseased pigs mainly manifest as white or off-white loose stools, and the incidence rate can reach about 65%. In addition to causing the death of piglets, the disease...

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Problems solved by technology

These vaccines can effectively reduce the clinical morbidity, but because the cell wall of the inactivated vaccine contains lipopolysaccharide (LPS), it may cause certain side effects on the vaccinated animals, such as: mild fever, sows do not eat after vaccination, and the inoculation site There are inflammatory reactions, etc.; in addition, the preparation process of adhesin vaccines, enterotoxin vaccines and genetic engineering vaccines is complicated and the price is high; 2. Biological prevention: Fang Dingyi and Dong Guoxiong (1978, 1979) once obtained from piglet intestinal tract A strain of Escherichia coli - NY-10 was isolated, which is a non-pathogenic Escherichia coli that can effectively repel pathogenic Escherichia coli in the intestinal tract. Clinical trials have shown that NY-10 can complete the intestinal tract within 24 hours. The colonization of the tract, and accounted for an overwhelming advantage, duration of 7 days, after oral administration can effectively prevent the occurrence of diarrhea in piglets, improve the survival rate of piglets (Fang Dingyi, Dong Guoxiong. Use non-pathogenic Ny-10 strains of large intestine Bacillus biological control of yellow scours in piglets. Jiangsu Agricultural Science and Technology, 1978, 5 (13): 55-58. Fang Dingyi, Dong Guoxiong. Prevention of yellow scours in piglets by feeding NY-10 strains of Escherichia coli by mouth Research. Animal Husbandry and Veterinary Journal, 1979,10(2):79-84.), but the research background of this strain is not very clear, what type of enterotoxin it is, whether there is antibiotic resistance gene in the bacteria is not clear, and The bacterium's aseptic pili have poor adhesion, and the colonization time in the body is not long, so it is almost no longer used clinically; 3. Drug treatment: In theory, antibiotics are an effective drug for treating bacterial diseases, but due to the long-term use of antibiotics in clinical Indiscriminate use, abuse, and the use of antibiotics as additives or health-care drugs in feed have led to increasingly serious drug resistance of Escherichia coli. Studies have shown that Escherichia coli in piglets with yellow and white scours is resistant to tetracycline, ampicillin, amoxicillin, doxycycline, and compound new drugs. The drug resistance of commonly used antibiotics such as Promin, Trimethoprim, Cephradine, and Ciprofloxacin is over 86%, and a strain of bacteria has multiple drug-resistant spectrums. These current situations lead to poor clinical antibiotic treatment effect

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