CN109402035A - A kind of parenteral enteropathogenic E. Coli recombinant bacterial strain and its application - Google Patents

A kind of parenteral enteropathogenic E. Coli recombinant bacterial strain and its application Download PDF

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CN109402035A
CN109402035A CN201811314587.8A CN201811314587A CN109402035A CN 109402035 A CN109402035 A CN 109402035A CN 201811314587 A CN201811314587 A CN 201811314587A CN 109402035 A CN109402035 A CN 109402035A
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bacterial strain
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albumen
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谭臣
张焱焱
陈焕春
王湘如
宗冰冰
周红波
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Huazhong Agricultural University
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Abstract

The invention discloses a kind of parenteral enteropathogenic E. Coli recombinant bacterial strain and its applications, belong to microorganism and immunological technique field.Parenteral enteropathogenic E. Coli recombinant bacterial strain of the invention is while lacking kpsM gene and iucB gene and can be overexpressed the Escherichia coli PCN033 of OmpC albumen or OmpF albumen.Parenteral enteropathogenic E. Coli recombinant bacterial strain of the invention, while kpsM gene and iucB gene have been lacked, virulence greatly reduces compared with parent bacterium, high using it as vaccine safety；It can be overexpressed OmpC albumen or OmpF albumen, reach 100% to the immune protective rate of mouse, have good application value.

Description

A kind of parenteral enteropathogenic E. Coli recombinant bacterial strain and its application
Technical field
The present invention relates to microorganisms and immunological technique field, and in particular to a kind of parenteral enteropathogenic E. Coli recombinant bacterial strain And its application.
Background technique
Escherichia coli is commonly called as Escherichia coli, is a kind of widely distributed Gram-negative bacteria in nature.According to it Pathogenic and pathogenesis difference, Escherichia coli are divided into symbiotic type Escherichia coli (Commensal Escherichia Coli), enteropathogenic Escherichia coli (Intestinal pathogenic Escherichia coli, IPEC) and parenteral Enteropathogenic E. Coli (Extraintestinal pathogenic Escherichia coli, ExPEC).
Parenteral enteropathogenic E. Coli can colonize tight in organizing and causing outside a variety of host intestines such as humans and animals extensively The infection of weight, such as urinary system infection contamination, meningitis, septicemia, pneumonia, peritonitis are different from traditional symbiotic type large intestine Bacillus and classical enteropathogenic Escherichia coli have been found to seriously endanger domestic animal, poultry and mankind's publilc health A kind of Zoonosis Escherichia coli, and medical field and sphere of learning report and pay close attention to that more one kind is important to cause a disease in recent years Bacterium.On veterinary clinic, agricultural microorganism National Key Laboratory, Hua Zhong Agriculture University is since 2004 persistently to national different provinces City, the morbidity pig farm in area and inspection pathological material of disease carry out Pathogen test with separate identification, from morbidity or death parenteral group of swinery It knits and isolates nearly thousand plants of ExPEC bacterial strains in (lung, brain, kidney, lymph node, joint, blood etc.), preliminary epidemiology statistics are aobvious Show, ExPEC, which has become, seriously threatens one of great bacterial pathogen of China's pig breeding industry.At the same time, ExPEC resistant strains Phenomenon is universal, and serious with other cause of disease mixed infections, causes huge loss to pig breeding industry, and increase high epidemic disease Prevention and control cost.It can be said that causing the related epidemic disease of different hosts to have become by ExPEC seriously affects human health, food peace The important infectious diseases common to human beings and animals of one kind of complete and animal husbandry development.At present for clinically popular wide and high disease incidence intestines There is no commercialized vaccines can be used for outer enteropathogenic E. Coli.
Summary of the invention
It is of the existing technology the purpose of the present invention is to provide solving the problems, such as, it provides a kind of less toxic, highly-safe, immune The good parenteral enteropathogenic E. Coli recombinant bacterial strain of protecting effect and its application.
The purpose of the invention is achieved by the following technical solution:
A kind of parenteral enteropathogenic E. Coli recombinant bacterial strain is while lacking kpsM gene and iucB gene and can be overexpressed The Escherichia coli PCN033 of OmpC albumen or OmpF albumen.Wherein, OmpC albumen, OmpF albumen amino acid sequence respectively such as SEQ ID NO.1, shown in 2.
The construction method of the parenteral enteropathogenic E. Coli recombinant bacterial strain, includes the following steps:
(1) genetic fragment for encoding OmpC albumen or OmpF albumen is connected on expression vector, constructs recombinant vector.Institute The expression vector stated is preferably PHSG396.
(2) recombinant vector is transferred to while is lacked in the Escherichia coli PCN033 of kpsM gene and iucB gene, obtained Parenteral enteropathogenic E. Coli recombinant bacterial strain.Wherein, it while lacking the Escherichia coli PCN033 of kpsM gene and iucB gene and is PCN033 Δ kpsM- Δ iucB, building are detailed in " the outer enteropathogenic E. Coli separation identification of chitling and dual-gene deletion mutation strain Building " [academic dissertation] Zhang Ru 2013- Hua Zhong Agriculture University: animal doctor.
The parenteral enteropathogenic E. Coli recombinant bacterial strain virulence is low, and immune protective effect is good, can be used for preparing prevention and control The vaccine of parenteral enteropathogenic E. Coli.
A kind of bacillus coli vaccine can also include its medicine comprising the parenteral enteropathogenic E. Coli recombinant bacterial strain Acceptable adjuvant on.
The present invention, then will be outer on the basis of the dual-gene gene-deleted strain PCN033 Δ kpsM- Δ iucB of constructed weak poison Memebrane protein ompC or ompF genetic recombination enters the low virulent strain, obtain a kind of immunoprotection it is high-efficient and safe can be used for preparing epidemic disease The recombinant bacterial strain of seedling.The invention has the advantages that and the utility model has the advantages that parenteral enteropathogenic E. Coli recombinant bacterial strain of the invention, KpsM gene and iucB gene are lacked simultaneously, virulence greatly reduces compared with parent bacterium, using it as vaccine safety It is ensured；It can be overexpressed OmpC albumen or OmpF albumen, reached 100% to the immune protective rate of mouse, had Good application value.
Detailed description of the invention
Fig. 1 is the PCR qualification figure of over-express vector PHSG396-ompC and PHSG396-ompF.In figure, M:DNA Ladder(DL2000)；1:PHSG396-ompC；2: negative control；3:PHSG396-ompF；4: negative control.
Fig. 2 is the double digestion qualification figure of over-express vector PHSG396-ompC and PHSG396-ompF.In figure, M1:DNA Ladder(DL2000)；M2:DNALadder (DL15000)；1:PHSG396-ompC；2:PHSG396-ompF；3:PHSG396.
Fig. 3 is the PCR of recombinant bacterial strain PCN033 Δ kpsM- Δ iucB+OmpC and PCN033 Δ kpsM- Δ iucB+OmpF Qualification figure.In figure, M:DNA Ladder (DL2000)；1:PCN033 Δ kpsM- Δ iucB+OmpC；2: negative control；3: PCN033ΔkpsM-ΔiucB+OmpF；4: negative control.
Specific embodiment
Following embodiment should not be construed as limiting the invention for further illustrating the present invention.If not referring in particular to Conventional means bright, that technological means used in embodiment is well known to those skilled in the art.
Used dual-gene gene-deleted strain PCN033 Δ kpsM- Δ iucB in following embodiments, with parenteral pathogenic large intestine Bacillus PCN033 is parent strain, while having lacked kpsM gene and iucB gene.Dual-gene gene-deleted strain PCN033 Δ kpsM- Δ The building of iucB is detailed in " the outer enteropathogenic E. Coli separation identification of chitling and dual-gene deletion mutation strain building " [academic dissertation] Zhang Ru 2013- Hua Zhong Agriculture University: animal doctor.The expression and purifying of OmpC and OmpF albumen used are detailed in that " pig source is parenteral pathogenic The research of Escherichia coli comparative genomics and subunit vaccine " [academic dissertation] Liu Can grain husk 2013- Hua Zhong Agriculture University: beast Doctor.
The building of embodiment 1 over-express vector PHSG396-ompC and PHSG396-ompF
Using the full-length genome of parenteral enteropathogenic E. Coli as template, expanding ompC gene respectively by PCR, (sequence is such as Shown in SEQ ID NO.3) and ompF gene (sequence is as shown in SEQ ID NO.4), PCR amplification the primer is respectively as follows:
OmpC1:ATCGGAGCTCATGAAAGTTAAAGTACTGTCCCTCC,
OmpC2:GGCGTCTAGATTAGAACTGGTAAACCAGRCCMA, ompC gene size 1104bp；
OmpF1:ATCGGAGCTCATGATGAAGCGCAATATTCT,
OmpF2:GGCGTCTAGATTAGAACTGGTAAACGATAC, ompF gene size 1095bp.
PCR reaction condition are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30sec, 56 DEG C of annealing 30sec, 72 DEG C extend 10min, denaturation are arrived anneal cycles 30 times；72 DEG C of extension 10min.
It, by glue recovery purifying, will will be purified after pcr amplification product and over-express vector PHSG396 difference double digestion processing OmpC and ompF segment connect overnight with the over-express vector PHSG396 of purifying in 4 DEG C respectively.By connection product electrotransformation sense By state DH5 α, it is coated on the solid plate containing chloramphenicol after recovery 1h and cultivates 12h, PCR is passed through to the single colonie of growth Whether identification recombinant plasmid constructs success, identifies primer with above-mentioned amplimer, and qualification result is shown in Fig. 1, institute's amplified fragments with OmpC gene is consistent with ompF gene size.After the single colonie of identification is accessed fluid nutrient medium culture, then extract plasmid use Sac I, Xba I carry out double digestion identification, as a result see Fig. 2, the small fragment that digestion is got off and ompC gene and ompF gene size one It causes, shows that over-express vector PHSG396-ompC and PHSG396-ompF are constructed successfully.
The structure of 2 recombinant bacterial strain PCN033 Δ kpsM- Δ iucB+OmpC and PCN033 Δ kpsM- Δ iucB+OmpF of embodiment It builds
By over-express vector PHSG396-ompC and PHSG396-ompF that embodiment 1 constructs, electricity is transferred to dual-gene lack respectively Strain PCN033 Δ kpsM- Δ iucB is lost, electricity is turned into liquid and is transferred in sterilizing EP pipe, 900 μ L LB culture solutions are separately added into, After cultivating 1h in 37 DEG C of shaking tables, it is coated on the solid culture plate containing chloramphenicol and cultivates 12h.On random picking plate Single colonie carries out PCR identification, and PCR primer is the same as embodiment 1.As a result see that Fig. 3, institute's amplified fragments and ompC gene and ompF gene are big It is small consistent, show that over-express vector PHSG396-ompC and PHSG396-ompF are successfully transferred to dual-gene gene-deleted strain respectively In PCN033 Δ kpsM- Δ iucB, the recombinant bacterial strain PCN033 Δ kpsM- Δ of OmpC albumen, ompF albumen can be overexpressed IucB+OmpC, PCN033 Δ kpsM- Δ iucB+OmpF are constructed successfully.
3 recombinant bacterial strain PCN033 Δ kpsM- Δ iucB+OmpC and PCN033 Δ kpsM- Δ iucB+OmpF's of embodiment exempts from Epidemic disease Protection
48 four week old kunming mices are taken, mouse is equally divided into 8 groups, is numbered respectively.
Wherein 1-7 group is experimental group (each 6), and the 8th group is attacked malicious group (6) for blank.
The OmpC albumen of 1st group of 50 μ g of immunity inoculation purifying: albumen is dissolved in 200 μ L PBS, 200 μ L concentration are added It mixes for 33% aluminium hydroxide for immunity inoculation.
The OmpF albumen of 2nd group of 50 μ g of immunity inoculation purifying: albumen is dissolved in 200 μ L PBS, 200 μ L concentration are added It mixes for 33% aluminium hydroxide for immunity inoculation.
The OmpF albumen of OmpC albumen and 25 μ g purifying that 3rd group of 25 μ g of immunity inoculation is purified: albumen is dissolved in 200 μ L In PBS, adds the aluminium hydroxide that 200 μ L concentration are 33% and mix for immunity inoculation.
4th group of immunity inoculation 1 × 108The dual-gene gene-deleted strain PCN033 Δ kpsM- Δ iucB of CFU: with PBS by PCN033 Δ It is 1 × 10 that kpsM- Δ iucB, which is diluted to concentration,9The bacterium solution of CFU/mL takes 100 μ L for immunity inoculation.
5th group of immunity inoculation 1 × 108CFU recombinant bacterial strain PCN033 Δ kpsM- Δ iucB+OmpC: with PBS by PCN033 It is 1 × 10 that Δ kpsM- Δ iucB+OmpC, which is diluted to concentration,9The bacterium solution of CFU/mL takes 100 μ L for immunity inoculation.
6th group of immunity inoculation 1 × 108The recombinant bacterial strain PCN033 Δ kpsM- Δ iucB+OmpF of CFU: will with PBS It is 1 × 10 that PCN033 Δ kpsM- Δ iucB+OmpF, which is diluted to concentration,9The bacterium solution of CFU/mL takes 100 μ L for immunity inoculation.
7th group of immunity inoculation 5 × 107The recombinant bacterial strain PCN033 Δ kpsM- Δ iucB+OmpC and 5 × 10 of CFU7CFU's Recombinant bacterial strain PCN033 Δ kpsM- Δ iucB+OmpF: with PBS by PCN033 Δ kpsM- Δ iucB+OmpC, PCN033 Δ KpsM- Δ iucB+OmpF is diluted to concentration 1 × 109The bacterium solution of CFU/mL respectively takes 50 μ L to mix for immunity inoculation.
1) first time immunity inoculation is carried out after a week to the raising of four week old mouse, 1-7 group is corresponding in subcutaneous immunizations Antigen, the 8th group of immunity inoculation PBS+ aluminium hydroxide (200 μ L PBS+200 μ L, 33% aluminium hydroxide)；
2) second of immunity inoculation is carried out after immune 14 days for the first time, 1-7 group is in the identical antigen of subcutaneous immunizations, and the 8 groups of immunity inoculation PBS+ aluminium hydroxides；
3) poison is attacked after being immunized 14 days for the second time, 1-8 group mouse peritoneal injects 100 μ L 1.5 × 10 respectively7The wild mushroom of CFU PCN033 bacterium solution.
The mouse after poison of attacking against each other is observed continuously 7 days, records every group of mouse survival rate.As a result (table 1) is shown: being attacked by abdominal cavity Poison injection 1.5 × 107CFU PCN033 bacterium solution, it is all dead for 24 hours in the dust about 50% in the Kunming mouse 12h of the 8th group of control group It dies；4th group of mouse death rate of immunity inoculation PCN033 Δ kpsM- Δ iucB antigen reaches 33%, immunity inoculation PCN033 Δ 5th, 6 group of mouse 100% of kpsM- Δ iucB+OmpC, PCN033 Δ kpsM- Δ iucB+OmpF antigen survives.Immunity inoculation The 1st group of antigen OmpC reaches 33% in the death rate for 24 hours；The 2nd group of immunity inoculation antigen OmpF, reaches in the 36h death rate 33%；The 3rd group of immunity inoculation antigen OmpC and OmpF reaches 33% in the 60h death rate.
Table 1 is immunized, attack poison after be observed continuously 7 days, the survival condition of every group of mouse
The above results show recombinant bacterial strain PCN033 Δ IuB-KpsM+OmpC and PCN033 Δ IuB-KpsM+OmpF to small The immune protective effect of mouse is significantly higher than albumen OmpC and OmpF to the immune protective effect of mouse, can be used for preparing prevention and control parenteral The vaccine of enteropathogenic E. Coli.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
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ggtggtgata ctgcatacag cgatgacttc ttcgttggtc gtgttggcgg cgttgctacc 480
tatcgtaact ccaacttctt tggtctggtt gatggcctga acttcgctgt tcagtacctg 540
ggtaaaaacg agcgtgacac tgcacgccgt tctaacggcg acggtgttgg cggttctatc 600
agctacgaat acgaaggctt tggtatcgtt ggtgcttatg gtgcagctga ccgtaccaac 660
ctgcaagaag ctcaacctct tggcaacggt aaaaaagctg aacagtgggc tactggtctg 720
aagtacgacg cgaacaacat ctacctggca gcgaactacg gtgaaacccg taacgctacg 780
ccgatcacta ataaatttac aaacaccagc ggcttcgcca acaaaacgca agacgttctg 840
ttagttgcgc aataccagtt cgatttcggt ctgcgtccgt ccatcgctta caccaaatct 900
aaagcgaaag acgtagaagg tatcggtgat gttgatctgg tgaactactt tgaagtgggc 960
gcaacctact acttcaacaa aaacatgtcc acctatgttg actacatcat caaccagatc 1020
gattctgaca acaaactggg cgtaggttca gacgacaccg ttgctgtggg tatcgtttac 1080
cagttctaa 1089

Claims (7)

1. a kind of parenteral enteropathogenic E. Coli recombinant bacterial strain, it is characterised in that: the recombinant bacterial strain is while lacking kpsM base Cause and iucB gene and the Escherichia coli PCN033 that OmpC albumen or OmpF albumen can be overexpressed.
2. parenteral enteropathogenic E. Coli recombinant bacterial strain according to claim 1, it is characterised in that: the OmpC egg White, OmpF albumen amino acid sequence is respectively as shown in SEQ ID NO.1,2.
3. the construction method of parenteral enteropathogenic E. Coli recombinant bacterial strain of any of claims 1 or 2, it is characterised in that: including Following steps:
(1) genetic fragment for encoding OmpC albumen or OmpF albumen is connected on expression vector, constructs recombinant vector；
(2) recombinant vector is transferred to while is lacked in the Escherichia coli PCN033 of kpsM gene and iucB gene, obtained parenteral Enteropathogenic E. Coli recombinant bacterial strain.
4. construction method according to claim 3, it is characterised in that: expression vector described in step (1) is PHSG396。
5. parenteral enteropathogenic E. Coli recombinant bacterial strain of any of claims 1 or 2 is preparing parenteral enteropathogenic E. Coli epidemic disease Application in seedling.
6. a kind of bacillus coli vaccine, it is characterised in that: include parenteral enteropathogenic E. Coli weight of any of claims 1 or 2 Group bacterial strain.
7. bacillus coli vaccine according to claim 6, it is characterised in that: include its pharmaceutically acceptable adjuvant.
CN201811314587.8A 2018-11-06 2018-11-06 A kind of parenteral enteropathogenic E. Coli recombinant bacterial strain and its application Pending CN109402035A (en)

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