CN106754535B - Recombinant salmonella enteritidis with weak toxicity - Google Patents
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Abstract
The invention provides a Salmonella attenuated enteritis rSD strain with the preservation number of CGMCC NO. 13346. The attenuated salmonella enteritidis provided by the invention is used for preparing a recombinant vaccine strain. In another aspect, the present invention provides a recombinant salmonella enteritidis live vector vaccine strain, the genome of which comprises VP2 gene of IBDV. The preservation number of one recombinant salmonella enteritidis live vector vaccine strain is CGMCC NO. 13251. The recombinant vaccine strain constructed by taking the salmonella enteritidis as a host has good genetic stability, and does not generate any mutation in the inserted exogenous gene after continuous passage for 20 generations in a common culture medium; the recombinant bacteria do not contain any resistance, which accords with the development trend of modern bacterial live vaccines, and has good safety without considering antibiotic residues. The recombinant bacteria have good growth and reproduction performance, and the multiplication of the bacteria is not influenced by the insertion of foreign genes.
Description
Technical Field
The invention belongs to the technical field of biological products for livestock, and particularly relates to recombinant salmonella enteritidis with weak toxicity.
Technical Field
Infectious Bursal Disease (IBD) is a highly contagious disease of chickens and turkeys caused by Infectious Bursal Disease Virus (IBDV). High incidence rate and short course of disease. The disease mainly attacks chicks and young chickens of 3-12 weeks old, destroys B lymphocytes of bursa of Fabricius, causes immunosuppression of different degrees, and can induce various epidemic diseases or cause immunity failure of various vaccines, so that the susceptibility of the sick chickens to concurrent and secondary virus and bacterial infection is increased, and the disease is one of important infectious diseases seriously threatening the poultry industry in China in recent years.
Currently, in the prevention of IBD, conventional live vaccines and inactivated vaccines are mainly used. The traditional inactivated vaccine goes through a process from a low-virulent vaccine to a high-virulent vaccine and from a single-strain vaccine to a multi-strain vaccine. Although IBD antibody titer of chicken can be improved by developing a vaccine by using a mesogenic or velogenic strain, the target organ bursa of Fabricius tissue is easily damaged, so that the immune function is low, the immunity is inhibited, the susceptibility to other diseases is increased, or the immune response capability to other vaccines is reduced, and other diseases are easy to secondary; the cross protection effect of the type I vaccine virus on the super-strong virus and the variant strain which are both type I is neglected theoretically, the possibility of recombination of different strains when the strains are proliferated in the same target cell is neglected, the harm to ecological safety caused by the recombination is neglected, and the error regions are avoided in the research and development of the vaccine. The inactivated vaccine is mostly prepared from chick embryos or cell adaptive virus, the cost is high, and the use of the inactivated vaccine is restricted. Therefore, the development of novel vaccines with broad spectrum, high efficiency and practicability is urgently needed.
In the research and application of novel vaccines, the immune protection effect of directly applying the cloned and expressed antigen protein as the vaccine is not ideal. This is because soluble antigens in the novel vaccines tend to be less immunogenic and do not provoke effective mucosal immunity. The most successful strategy to solve this problem is to use vaccine vectors, in which salmonella has gained extensive research and use by its own advantages, to deliver antigens to mucosal surfaces. However, the existing method has the problems that a stable salmonella low virulent strain is lacked, and exogenous protein cannot be stably expressed, so that effective immune response is caused, and the like.
Disclosure of Invention
The invention aims to provide a low-virulent salmonella enteritidis and a recombinant salmonella enteritidis low-virulent strain constructed by taking the low-virulent salmonella enteritidis as a host, and the low-virulent strain can be used for preparing vaccines.
The invention firstly provides a Salmonella Enteritidis (Salmonella Enteritidis) rSD strain which is preserved in China general microbiological culture Collection center in 2016, 11 and 28 days, and the addresses are as follows: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences. The preservation number is: CGMCC NO. 13346.
The attenuated salmonella enteritidis provided by the invention is used for preparing a recombinant vaccine strain;
the recombinant vaccine strain is prepared by transferring exogenous immune antigen genes into the salmonella enteritidis;
preferably, the immune antigen gene is VP2 gene of IBDV;
in another aspect, the present invention provides a recombinant salmonella enteritidis live vector vaccine strain, the genome of which comprises VP2 gene of IBDV.
The recombinant salmonella enteritidis live vector vaccine strain is named as salmonella enteritidis △ VP2(salmonella), and is preserved in China general microbiological culture Collection center (CGMCC) in 2016, 11, 10 and 11 months, with the address of No. 3 Beijing university Hopkin No.1 Hopkin, West Lu of the Korean district, China academy of sciences microbial research institute, and the preservation number of CGMCC NO. 13251.
The recombinant salmonella live vector vaccine strain provided by the invention can be used for preparing vaccines.
The recombinant vaccine strain constructed by taking the salmonella enteritidis as a host has good genetic stability, and does not generate any mutation in the inserted exogenous gene after continuous passage for 20 generations in a common culture medium; the recombinant bacteria do not contain any resistance, which accords with the development trend of modern bacterial live vaccines, and has good safety without considering antibiotic residues. The recombinant bacteria have good growth and reproduction performance, and the multiplication of the bacteria is not influenced by the insertion of foreign genes.
Drawings
FIG. 1 shows the growth curves of wild strain SD, attenuated strain rSD and recombinant strain △ VP2 (salmonella).
Detailed Description
The salmonella enteritidis low virulent strain is obtained by screening, the low virulent strain is used for constructing a low virulent salmonella vaccine strain, the constructed recombinant low virulent salmonella vaccine strain is weak in virulence, good in safety, stable and high in expression efficiency, and the induced antibody can effectively provide immune protection; thus leading to the present invention.
Example 1 screening of Salmonella enteritidis attenuated strains
The applicant determines the suspected salmonella separated from the clinic to be salmonella enteritidis after biochemical test, serological test and sequencing identification, and the suspected salmonella is named as SD strain. After passage screening, the recombinant strain rSD strain of the salmonella enteritidis is obtained. The growth speed of bacteria is not influenced after gene deletion is found through the measurement of a growth curve, and the bacteria are determined to be low virulent strains through a chick challenge test and the like. The rSD strain was used as the parent strain for the next study.
The attenuated enteritis salmonella rSD strain is preserved in the China general microbiological culture Collection center in 2016, 11 and 28 months, and the addresses are as follows: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences. The preservation number is: CGMCC NO. 13346.
Example 2 construction of VP2 Gene transfer vector
Based on the sequence of the Cat gene in plasmid pkD3 and the polyclonal cleavage site of the cloning vector pBluescript KS (+/-), primers specific for the Cat gene were designed using Primer 5.0 software: the upstream primer pKD3-II P1: CACGGATCCgtgtaggctggagctgcttc is added with a BamH I enzyme cutting site; the downstream primer pKD3-II P2: CAGGAATTCcatatgaatatcctccttag adds an EcoR I enzyme cutting site. PCR amplification was performed using plasmid pkD3 as a template, and the objective gene was recovered using a DNA purification recovery kit. The recovered target gene fragment was subjected to double digestion reaction with the vector pBluescript KS (+/-), nucleic acid electrophoresis, recovery of the target gene using a DNA purification recovery kit, and ligation using T4DNA ligase to obtain a recombinant plasmid. The recombinant plasmid was transformed into Escherichia coli JM109, identified by sequencing and designated pBlu-pKD 3.
Amplifying VP2 gene by PCR, and carrying out upstream primer: VP 2-P1: CAGGAATTCATGACAAACCTGCAAGATCAAACCC, EcoR I enzyme cutting site is added, and the downstream primer: VP 2-P2: GACAAGCTTTTACCTTATGGCCCGGAT HindIII sites were added. Recovering the target gene, carrying out double enzyme digestion reaction with the plasmid pBlu-pKD3, carrying out 1% agarose gel electrophoresis, recovering the target gene by using a DNA purification recovery kit, and then connecting by using T4DNA ligase to obtain the recombinant plasmid. The recombinant plasmid was transformed into Escherichia coli JM109, sequenced, identified and named pBKV.
Example 3 preparation of targeting fragment
The primer for homologous recombination consists of two parts, wherein the sequence of 50nt with capital writing at the 5 'end is homologous with the flanking sequences of the target gene, and the sequence of 20nt with small writing at the 3' end is homologous with the sequences at both sides of the cat resistance gene or the target gene of the transfer vector. An upstream primer: VP 2-D1: AGTGGACTAACAACATCGGTGATGCCAACACCATCGGCACCCGTCCGGACgtgtaggctggagctgcttc, respectively; a downstream primer: VP 2-D2: CAGAGCAAAAAACCCCGCGACGCGGGGTTTTTTATCAGACGGAAACTTAAttaccttatggcccggat are provided. And (3) taking the recombinant plasmid pBKV as a template, amplifying a targeting fragment by PCR, identifying a PCR product by 1% agarose gel electrophoresis, recovering a target band, and digesting the recovered product by Dpn I. After electrophoresis on a 1% agarose gel, the gel was cut to recover the desired band, and the DNA concentration was determined.
Example 4 preparation of competent cells
Plasmid pKD46 is transformed into a virulent salmonella strain prepared by a calcium chloride method, the virulent salmonella strain is cultured on an ampicillin plate at 30 ℃ overnight, a single colony is selected to be inoculated on a L B culture medium containing aminobenzyl for shaking culture at 30 ℃ overnight, 400ul of culture is inoculated on a L B culture medium containing 10m L Amp, shaking culture is carried out at 220rpm until OD600 is 0.6, the bacteria are collected by ice bath for 10min, centrifugation is carried out at 4 ℃ and 4000rpm for 10min, precipitates are washed by 10% glycerol precooled by the ice bath for 3 times, and the precipitates are resuspended by 10% glycerol precooled by 40u L, thus obtaining competent cells.
Example 5 electroporation and Elimination of resistance genes
Mixing 2u L targeting fragment (about 100ng) with 40u L competent cells, transferring into 1mm BioRad electrode cup, ice-bath for 2min, electric shocking at 2KV for 5ms, immediately adding 1m L common L B culture medium, transferring into 15m L test tube, shaking at 120rpm and 37 deg.C for 3h, spreading 200u L on L B plate containing chloramphenicol (Cm), culturing at 37 deg.C overnight, and screening CmRSingle clone is picked up and cultured at 43 ℃, and Amp antibiotic test is carried out to screen the strain with loss of the helper plasmid pKD46, so as to obtain a mutant strain with only chloramphenicol resistance, which is named △ V P2: Cat (salmonella).
Plasmid pCP20 was electrically transferred to △ V P2: Cat (salmonella), 200. mu.l of the plasmid was spread on a L B plate containing Amp and Cm dual resistance, and positive transformants were selected by culturing at 30 ℃ and selecting a single clone, and a non-resistant recombinant strain was selected and named △ V P2(Salm onella).
Example 6 measurement of growth Curve
And (2) selecting single colonies of the wild strain SD strain, the parent strain rSD strain and the recombinant strain, respectively inoculating the single colonies into a liquid L B culture medium, carrying out shake culture in a constant temperature shaking table at 37 ℃ overnight, regulating OD600 of the bacterial suspension to be 0.1 by using 10m L liquid L B, carrying out shake culture at 37 ℃ at 220rpm, measuring OD600 of the bacterial solution every 1h, continuously measuring for 8h, and drawing a bacterial growth curve.
The results showed no significant difference in growth rate between the wild strain, the recombinant strain and the parent strain, demonstrating that genetic engineering of the wild strain, insertion of the VP2 gene in the attenuated salmonella genome did not affect the growth of the bacteria (fig. 1).
Example 7 in vitro stability test
Selecting single clones of an attenuated strain rSD strain and a recombinant strain △ V P2(salmonella) strain, respectively inoculating the single clones into a 15m L test tube, carrying out shake culture at 37 ℃ for 12h, inoculating the culture into a common L B liquid according to the amount of 1:1000, carrying out continuous passage for 20 times, taking 400ul of the bacterial liquid after continuous passage for 20 generations, extracting DNA by a boiling lysis method, respectively amplifying the parts of the rSD strain with gene deletion, carrying out △ V P2(salmonella) strain with gene insertion and deletion parts, recovering a target band after 1% agarose gel electrophoresis, connecting with a pMD 19-T vector, carrying out identification and sequencing, wherein the sequencing results show that no gene reversion occurs in the parts of the deleted genes and no gene mutation and deletion occur in the parts of the inserted target genes.
Example 8 in vitro proliferation assay of bacteria
Three strains of bacteria (SD strain, rSD strain and △ V P2(salmonella) strain), selecting single clone, inoculating into 15m L test tube, shake culturing at 37 deg.C for 12h, inoculating the above culture into 20m L common L B liquid culture medium at a ratio of 1:1000, culturing overnight, diluting 100u L bacterial liquid to 10 times-8Then respectively use 10-6、10-7And 10-8The bacterial liquid of three dilutions was counted by plating 100u L each, and the bacterial content was determined and repeated 3 times for each strain, and the results showed that the bacterial content of three overnight-cultured strains was about 8 × 10 per ml, which was not different from each other9CFU, which is consistent with the measurements of the growth curve.
Example 9 challenge test for wild strain SD, attenuated strain rSD and recombinant strain △ V P2(salmonella)
Three salmonella strains are statically cultured for 12h at 37 ℃ on L B plates, a single colony is selected and inoculated in a test tube with the diameter of 15m L, the bacteria are shaken overnight, and the concentration of the bacteria liquid is adjusted by a spectrophotometer.
1 day-old SPF chickens are randomly divided into 3 groups, each large group is a virus attacking group of three strains, each large group is divided into 4 small groups, wherein 3 small groups are virus attacking groups, 1 small group is a control group, each small group is 8, each small chicken in the virus attacking group takes 0.5m L bacterial liquid orally, and the bacterial liquid is divided into 1011CFU、1010CFU and 109Negative control group each chicken was orally administered 0.5m L liquid L b for two weeks of continuous observation.
The test results show that in the test groups of the attenuated rSD strain and the recombinant strain △ V P2(salmonella), the chicks do not die in the observation period of two weeks, adverse reactions such as diarrhea, disordered feathers, abnormal activities and the like do not occur, the states of the chickens in the challenge group and the control group are not different, and 10 inoculation steps are carried out on the wild strain SD strain test group11CFU、1010CFU and 109The CFU dose groups died 7, 4 and 3 chickens each, and the remaining surviving chickens grew poorly, were loose, thinned, etc., while the control group did not have any signs of death or morbidity.
EXAMPLE 10 chick Immunity test with recombinant Salmonella enteritidis △ V P2(salmonella) Strain
The 1-day-old SPF chickens were randomly divided into three groups, two groups of immunization groups, one group of negative control group, 8 bacteria in each group cultured overnight, the bacterial concentration was adjusted, and in the immunization groups, 0.5m L (10) was orally administered to each of the first group9CFU), a second group of attenuated salmonella rSD strains with the same oral dose, and a control group of attenuated salmonella rSD strains with the same oral dose, wherein blood is collected in one week, two weeks, three weeks and four weeks after immunization respectively, serum is separated, an agar diffusion test is adopted to measure an antibody, and an antigen is detected as prokaryotic expression VP2 protein.
The results show that the recombinant strain △ V P2(salmonella) can produce antibody after one week, 100% of chicks can produce antibody after two weeks, the antibody level gradually begins to decline after four weeks, and the attenuated strain rSD strain and L B immune groups can not produce antibody.
TABLE 1 recombinant strain △ V P2(salmonella) strain immunohistochemical antibody level monitoring
Example 11 immune challenge protection assay
The immunization method and dose were the same as in example 10, and 8 were used in both the two groups of the immunized group and the one group of the control group. 3 weeks after immunization the chickens were inoculated nasally, eyedrops with virulent IBDV WF strain isolated in the home chamber (10)5TCID50/0.2m L) control group and immunization group were kept in isolation, and the status of chicks was observed every day, and only dead chicks were anatomically observed.
The results show that L B and attenuated rSD strain immune groups begin to get ill on the third day of virus attack, feathers are disorganized, the food consumption is reduced, the animals do not want to move, and the milk is thin and loose, L B groups die 2 on the fourth day, 3 die on the fifth day and 2 die on the sixth day, the attenuated rSD strain immune groups die 2 on the fourth day, 3 die on the fifth day and 1 die on the sixth day, chickens with the immune recombinant strain △ V P2(salmonella) strain are normal and do not have diarrhea and death, the dissected bursa shows that the recombinant strain △ V P2(salmonella) immune groups have no swelling and bleeding phenomena in bursa of Fabricius, and other two groups of chickens have swelling and bleeding phenomena in bursa of Fabricius, and some purple grape-like pathological changes appear, and △ V P2(salmonella) can provide good protection effect on IBDV infection.
Claims (1)
1. The recombinant salmonella enteritidis vaccine strain is characterized in that the preservation number of the recombinant salmonella enteritidis vaccine strain is CGMCC NO. 13251.
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| CN101920011A (en) * | 2010-06-29 | 2010-12-22 | 西北农林科技大学 | Recombination attenuated salmonella typhimurium carrier vaccine of expression IBDV (Infectious Bursal Disease Virus) immunogenic gene and preparation method thereof |
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