CN102031262A - Construction method and application of faeG expressing gene engineering probiotic - Google Patents
Construction method and application of faeG expressing gene engineering probiotic Download PDFInfo
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Abstract
The invention discloses a construction method and application of a faeG expressing gene engineering probiotic. The method comprises the following steps of: according to the gene sequence and the expression vector plasmid characteristic of a disclosed F4 escherichia coli pilus adhesin faeG, designing a primer which contains a specific endonuclease site; performing polymerase chain reaction (PCR) amplification by taking deoxyribonucleic acid (DNA) of an enterotoxigenic escherichia coli plasmid as a template to obtain a faeG gene-containing segment, linking the segment with an expression vector plasmid pNZ8148 to obtain a recombinant plasmid pNZ8148-faeG, transferring the recombinant plasmid into lactobacillus or a galactococcus cell by using an electrotransformation method to obtain gene engineering probiotic which expresses enterotoxigenic escherichia coli pilus adhesin faeG, expressing under the induction of nisin and analyzing and proving a target gene by SDS-PAGE and Western blot so as to express correctly. A recombinant strain can be used for preventing colibacillus diarrhea of suckling pigs and weanling pigs.
Description
Technical field
The invention belongs to the animal and veterinary technical field, relate in particular to a kind of genetically engineered probiotic bacterium structure and application in prevention of coliform diarrhea in piglets thereof of producing enterotoxigenic escherichia coli adhesin gene faeG of expressing.
Background technology
By produce enterotoxigenic escherichia coli (Enterotoxigenic Escherichia coli, the grice diarrhoea that ETEC) causes constitutes a serious threat to Swine Production, therefore pig industry annual and suffer enormous economic loss.Coliform diarrhea in piglets comprises sucking piglets diarrhoea and diarrhea of weaned piglets.The sucking piglets coliform diarrhea is divided into yellow scour of piglet and dysentery characterized by white mucous stool again.Yellow scour of piglet is acute, the lethality transmissible disease that newborn piglet is often sent out, and mainly betides 7 ages in days with interior sucking pig, the sucking pig pilosity of 1～3 age in days, and the morbidity back just is a feature to arrange yellow or yellow-white rare.Swinery is in case morbidity spreads very soon, and the sickness rate in a brood of piglet can reach 50%～100%, and what mortality ratio had reaches 100%.Baby pig pujos blancos is mainly in the sucking piglets of 10～30 ages in days, has the paste sample of stench flavor just rare with ash discharge white, but serious draining sample rare just be feature.The coliform diarrhea pilosity is born in 1 week of wean back behind the weaned piglet, though symptom is so obvious during unlike birth, and the weight gain of piglets speed that often slows down, the change of other factors such as ablactation stress, food etc. all can increase the weight of the state of an illness.
After piglet infected ETEC, the ETEC pili combined with the specific receptors of mucous membrane of small intestine epithelium, makes ETEC settle down in small intestine, a large amount of breedings, produce heat-stable toxin (heat-stable enterotoxin, ST) or/and heat-labile toxin (heat-labile enterotoxin, LT).Enterotoxin is invaded in the intestinal epithelial cell, changes epithelial normal absorption and secretion, upsets the small intestine metabolism, makes water, ionogen enter small intestinal lumen in a large number, thereby causes violent diarrhoea.In this process, pili and enterotoxin are considered to main virulence factor, and wherein to combine with small intestine epithelium be the first step that infects to pili, also is morbific key link.The pili antigen type difference of different ETEC bacterial strains mainly contains F4 (K88), F5 (K99), F6 (987P) and F41 etc., and modal in China is K88.
For the prevention of sucking piglets coliform diarrhea, existing at present commercialization vaccine is available, mainly contains F4 (K88)-F5 (K99) divalence seedling and intestinal bacteria deactivation vaccine.With these vaccine immunity soies heavy in pig, meeting produce specific antibody in the sow body, and piglet obtains maternal antibody by sucking colostrum, thereby stops the product enterotoxigenic escherichia coli to be settled down in intestines, avoids eqpidemic disease to take place; But existing commercialization vaccine to weaned piglet after coliform diarrhea be difficult to prove effective.In addition, business-like intestinal bacteria deactivation vaccine or bacillus coli gene engineering seedling because of bacterial cell wall contains lipopolysaccharides (intracellular toxin) toxic component, can produce stronger side reaction during the inoculation animal.
The biosynthesizing of F4 pili is relevant with 10 protein protomers of FaeA-FaeJ at least with assembling.Wherein FaeG content is maximum, is the main subunit of forming pili, and is relevant with the adhesion characteristics of pili, is the main binding site of F4 acceptor.Bacterium lacticum and galactococcus are regarded as the microorganism of generally recognized as safe (GRAS), cell walls does not contain lipopolysaccharides (intracellular toxin) toxic component, with it is the genetically engineered probiotic bacterium that the recipient bacterium construction expression produces enterotoxigenic escherichia coli adhesin gene faeG, be used for prevention of coliform diarrhea in piglets, have bright prospects.Domestic research in this field at present still belongs to blank.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, construction process and the purposes of the genetically engineered probiotic bacterium of a kind of expression faeG (producing enterotoxigenic escherichia coli adhesin gene) are provided, and immunogenicity of the present invention is good, have adjuvant effect, Stability Analysis of Structures, side effect is little, preparation is easy.
The objective of the invention is to be achieved through the following technical solutions: the construction process of the genetically engineered probiotic bacterium of a kind of faeG of expression, this method may further comprise the steps:
(1) obtain intestinal bacteria adhesin FaeG gene: to produce enterotoxigenic escherichia coli C83907 plasmid DNA is template, synthetic upstream and downstream primer P1 and the P2 that contains Nco I and Xba I restriction enzyme site of design, upstream and downstream primer P1 and P2 have the gene order of SEQ ID NO.4 and SEQ ID NO.5 respectively.Adopt PCR method amplification faeG target gene fragment.The faeG target gene fragment of amplification is connected with the pMD18-T carrier, gets recombinant plasmid, change among the intestinal bacteria TOP10, this recombinant plasmid called after pMD-faeG, it has the gene order shown in the SEQ ID NO.1.
(2) make up recombinant expression vector pNZ8148-faeG: with expression vector plasmid pNZ8148 and pMD-faeG restriction enzyme Nco I and Xba I double digestion, purifying reclaims double digestion carrier segments and purpose fragment.The purpose fragment is connected back transformed competence colibacillus E.coli MC1061 with the double digestion carrier segments, extract recombinant plasmid, called after pNZ8148-fae G, and it has the gene order shown in the SEQ ID NO.2.
(3) the recombination lactic acid galactococcus of construction expression intestinal bacteria adhesin gene faeG: adopt electric method for transformation with recombinant plasmid pNZ8148-faeG transformed competence colibacillus cell L.lactis NZ9000, utilize paraxin that transformant is screened, the bacterium of must recombinating, called after NZ9000/8148-faeG, it has the gene order shown in the SEQ ID NO.3.
(4) abduction delivering and SDS-PAGE, the Westernblot of intestinal bacteria adhesin FaeG in Lactococcus lactis analyzes: picking reorganization bacterium NZ9000/8148-faeG is inoculated in the GM17 liquid nutrient medium that contains 10mg/mL paraxin, and 30 ℃ leave standstill overnight incubation.Culture is transferred with 3% ratio and is contained in the paraxin GM17 nutrient solution in 500ml, is cultured to OD
600=0.5～0.6 o'clock, add inductor nisin, making its final concentration is 5ng/ml, continues to cultivate 3h.
The application of genetically engineered probiotic bacterium in prevention sucking piglets and weanling pig coliform diarrhea of enterotoxigenic escherichia coli adhesin gene faeG produced in a kind of above-mentioned expression.
The invention has the beneficial effects as follows: genetically engineered recipient bacterium Bacterium lacticum and galactococcus are generally regarded as safe microorganisms, do not contain intracellular toxin, do not secrete extracellular toxin, as vaccine carrier to target animals and human security; After genetically engineered Bacterium lacticum that makes up and galactococcus are expressed purpose antigen, do not need can directly to use through complicated last handling process such as purification, renaturation, vaccine production is easy; Bacterium lacticum and galactococcus self have adjuvant effect, can strengthen the immune effect of expressed purpose antigen (Fae G); When using as oral vaccine, Bacterium lacticum and galactococcus thalline shield to expressed purpose antigen (Fae G), and the degraded of opposing gi tract adverse circumstance destroys, and improves immune effect.
Description of drawings
Fig. 1 is an expression vector plasmid pNZ8148 collection of illustrative plates;
Fig. 2 is a pMD-fae G recombinant plasmid double digestion product electrophoretogram, and among the figure, 1 is DNA MarkerDL2, and 000,2 is the fragment of recombinant plasmid through NcoI and the generation of XbaI double digestion;
Fig. 3 is that recombinant plasmid pNZ8148-faeG electricity transforms galactococcus PCR evaluation electrophoretogram, and among the figure, 1 is DL2,000DNA Marker, and 2-10 is reorganization bacterium PCR product;
Fig. 4 is that recombinant plasmid pNZ8148-faeG electricity transforms galactococcus extraction plasmid enzyme restriction product electrophoretogram, and among the figure, 1 is DL2,000DNA Marker, and 2-4 extracts the plasmid enzyme restriction product for the reorganization bacterium;
Fig. 5 is the SDS-PAGE electrophoretogram of reorganization bacterium expression product, and among the figure, M is the protein standard molecular weight, and 1 for containing blank plasmid pNZ8148 galactococcus, and 2 for containing recombinant plasmid pNZ8148-faeG galactococcus;
Fig. 6 is the Western-blotting collection of illustrative plates of reorganization bacterium expression product, 1 is intestinal bacteria C83907 (adhesin gene faeG is used to increase), 2 is the protein standard molecular weight, and 3-4 is for containing blank plasmid pNZ8148 galactococcus, and 5 for containing recombinant plasmid pNZ8148-faeG galactococcus;
Fig. 7 is the dynamic change of reorganization bacterial immunity mice serum FaeG specific IgG;
Fig. 8 is reorganization fungus oral immune mouse excrement FaeG specificity SIgA dynamic change.
Embodiment
The recombinant probiotics that enterotoxigenic escherichia coli adhesin gene faeG is produced in expression of the present invention adopts following method to make up: according to gene order and the expression vector plasmid characteristics of disclosed F4 coli common pili adhesin FaeG, design contains the primer of special restriction enzyme site; To separate the product enterotoxigenic escherichia coli plasmid DNA that obtains or buy from the diarrhoea piglet is template, carry out pcr amplification, acquisition contains the fragment of faeG gene, it is connected with expression vector plasmid pNZ8148, obtain recombinant plasmid pNZ8148-faeG, by electric method for transformation this recombinant plasmid is changed in Bacterium lacticum or the galactococcus cell, under the inducing of nisin (nisin), express.
Wherein the F4 intestinal bacteria comprise F4ab, F4ac and 3 serotypes of F4ad; The expression vector plasmid is pNZ8148 or is that the basis changes the plasmid that obtains after the selective marker with pNZ8148; Recipient bacterium is Bacterium lacticum or the galactococcus that is integrated with nisK and nisR gene on the karyomit(e); Be used to increase the gene order of F4 coli common pili adhesin gene faeG segmental upstream and downstream primer P1 and P2 shown in SEQ ID NO.4 and SEQ IDNO.5:
P1：
CCATGGGGATGACTGGTGATTTCAATG
Nco?I
P2：
TCTAGAATAAGTAATTGCTACGTTCAG
Xba?I
Bacterium lacticum and galactococcus are regarded as the microorganism of generally recognized as safe (GRAS), belong to gram-positive microorganism, cell walls does not contain lipopolysaccharides (intracellular toxin) toxic component, and have multiple effects such as the growth of promotion animal digestive tract probiotics, the harmful bacterium breeding of inhibition, enhancing animal immune function, be applied in the Production of Livestock and Poultry as probiotic feed additive.The recombinant probiotics of enterotoxigenic escherichia coli adhesin gene faeG is produced in the expression that the present invention makes up; be used for the coliform diarrhea in piglets prevention as vaccine; not only have antigen protection, slow releasing function but also have adjuvant effect concurrently, can obtain than existing based on the better effect of the conventional vaccine of intestinal bacteria.Concrete application method is as follows:
The sucking piglets coliform diarrhea: with the antenatal immune sow of injecting pathway, piglet prevents coliform diarrhea lactation by sucking colostrum acquisition maternal antibody to the recombinant probiotics of expression product enterotoxigenic escherichia coli adhesin gene faeG after deactivation.
Weanling pig coliform diarrhea: express the recombinant probiotics that produces enterotoxigenic escherichia coli adhesin gene faeG and inoculate piglet with oral route, by activating mucosal immune response, produce adhesin FaeG specificity SIgA, combine prevention weanling pig coliform diarrhea with the pig intestinal mucosa epithelial receptor thereby combine the blocking-up germ with product enterotoxigenic escherichia coli pili.
Through the test of a series of immunoprotections, prove the effective prevention of coliform diarrhea in piglets of recombinant probiotics of expressing product enterotoxigenic escherichia coli adhesin gene faeG.
For example the present invention is done description in more detail below in conjunction with accompanying drawing, it is more obvious that purpose of the present invention and effect will become.
1, biomaterial
(1) target gene sequences
The gene order of intestinal bacteria adhesin FaeG comes among the Internet Genbank.
(2) vector plasmid
Expression vector plasmid pNZ8148 is from Dutch NIZO institute in the nisin induction type born of the same parents, and collection of illustrative plates sees Fig. 1 for details.
The pMD18-T carrier is available from Shanghai biotechnology Services Co., Ltd.
(3) bacterial strain
Intestinal bacteria TOP10 is available from Invitrogen company; Intestinal bacteria E.coli MC1061 is available from Invitrogen company; (O149:K91 K88ac) supervises institute from Chinese animal doctor to contain the product enterotoxigenic escherichia coli C83907 of intestinal bacteria adhesin FaeG gene; Recipient bacterium L.lactis NZ9000 is from Dutch NIZO institute.
2, the acquisition of intestinal bacteria adhesin FaeG gene
To produce enterotoxigenic escherichia coli C83907 plasmid DNA is template, and synthetic upstream and downstream primer P1 and the P2 that contains Nco I and Xba I restriction enzyme site of design adopts PCR method amplification faeG target gene fragment.FaeG gene PCR product is connected with the pMD18-T carrier, changes among the intestinal bacteria TOP10.Recombinant plasmid is cut evaluation (seeing Fig. 2 for details) and order-checking (the handsome Bioisystech Co., Ltd in Shanghai) analysis through enzyme, proves that the purpose fragment of amplification is the complete genome of faeG, this recombinant plasmid called after pMD-faeG, and its gene order is shown in SEQ ID NO.1.
The gene order of upstream and downstream primer P1 and P2 is shown in SEQ ID NO.4 and SEQ ID NO.5.
3, the structure of recombinant expression vector pNZ8148-fae G
With expression vector plasmid pNZ8148 and pMD-faeG restriction enzyme Nco I and Xba I double digestion, purifying reclaims double digestion carrier segments and purpose fragment.The purpose fragment is connected back transformed competence colibacillus E.coli MC1061 with the double digestion carrier segments, extract recombinant plasmid called after pNZ8148-faeG after enzyme is cut evaluation and order-checking (the handsome Bioisystech Co., Ltd in Shanghai) analysis verification, its gene order is shown in SEQ ID NO.2.
4, express the recombination lactic acid galactococcus structure of intestinal bacteria adhesin gene faeG
Adopt electric method for transformation with recombinant plasmid pNZ8148-faeG transformed competence colibacillus cell L.lactis NZ9000, the electric shock condition is voltage 2500V, resistance 400 Ω, electric capacity 25uF.Utilize paraxin that transformant is screened, identify the bacterium called after NZ9000/8148-faeG that recombinates after (seeing Fig. 3 for details), enzyme are cut evaluation (seeing Fig. 4 for details) and order-checking (the handsome Bioisystech Co., Ltd in Shanghai) checking through PCR, its gene order is shown in SEQ ID NO.3.
5, abduction delivering and SDS-PAGE, the Westernblot of intestinal bacteria adhesin FaeG in Lactococcus lactis analyzes
Picking reorganization bacterium NZ9000/8148-faeG is inoculated in the GM17 liquid nutrient medium that contains 10ug/mL paraxin, and 30 ℃ leave standstill overnight incubation.Culture is transferred with 3% ratio and is contained in the paraxin GM17 nutrient solution in 500ml, is cultured to OD
600=0.5～0.6 o'clock, add inductor nisin, making its final concentration is 5ng/ml, continues to cultivate 3h.The reorganization bacterium cracking after SDS-PAGE (seeing Fig. 5 for details) and Western blot (seeing Fig. 6 for details) analytical proof goal gene correctly expressed.The reorganization bacterium specific proteins band occurs at 27.0kD place after nisin induces, with expect big or small consistent.Gel shows that through the thin layer scanning analysis target protein accounts for 10% of recipient bacterium total protein.
6, the immune effect of recombinant vaccine
The recombination lactic acid galactococcus of the expression intestinal bacteria adhesin FaeG that the present invention makes up, adopt subcutaneous injection and oral route immune mouse respectively, result's (seeing Fig. 7,8 for details) shows: five all serum FaeG specific IgGs were tired and are higher than 1000 after (1) injection inoculation was just exempted from, and just exempted from back seven all serum titers and were higher than 4000; (2) oral route is just exempted from seven weeks of back, and Fae G specificity sIgA level is significantly higher than control group in the mouse excrement.
Among Fig. 7,
Among Fig. 8,
Claims (2)
1. construction process of expressing the genetically engineered probiotic bacterium of faeG is characterized in that this method may further comprise the steps:
(1) obtain intestinal bacteria adhesin FaeG gene: to produce enterotoxigenic escherichia coli C83907 plasmid DNA is template, synthetic upstream and downstream primer P1 and the P2 that contains Nco I and Xba I restriction enzyme site of design, upstream and downstream primer P1 and P2 have the gene order of SEQ ID NO.4 and SEQ ID NO.5 respectively.Adopt PCR method amplification faeG target gene fragment.The faeG target gene fragment of amplification is connected with the pMD18-T carrier, gets recombinant plasmid, change among the intestinal bacteria TOP10, this recombinant plasmid called after pMD-faeG, it has the gene order shown in the SEQ ID NO.1.
(2) make up recombinant expression vector pNZ8148-faeG: with expression vector plasmid pNZ8148 and pMD-faeG restriction enzyme Nco I and Xba I double digestion, purifying reclaims double digestion carrier segments and purpose fragment.The purpose fragment is connected back transformed competence colibacillus E.coli MC1061 with the double digestion carrier segments, extract recombinant plasmid, called after pNZ8148-fae G, and it has the gene order shown in the SEQ ID NO.2.
(3) the recombination lactic acid galactococcus of construction expression intestinal bacteria adhesin gene faeG: adopt electric method for transformation with recombinant plasmid pNZ8148-faeG transformed competence colibacillus cell L.lactis NZ9000, utilize paraxin that transformant is screened, the bacterium of must recombinating, called after NZ9000/8148-faeG, it has the gene order shown in the SEQ ID NO.3.
(4) abduction delivering and SDS-PAGE, the Westernblot of intestinal bacteria adhesin FaeG in Lactococcus lactis analyzes: picking reorganization bacterium NZ9000/8148-faeG is inoculated in the GM17 liquid nutrient medium that contains 10mg/mL paraxin, and 30 ℃ leave standstill overnight incubation.Culture is transferred with 3% ratio and is contained in the paraxin GM17 nutrient solution in 500ml, is cultured to OD
600=0.5～0.6 o'clock, add inductor nisin, making its final concentration is 5ng/ml, continues to cultivate 3h.
2. the application of genetically engineered probiotic bacterium in prevention sucking piglets and weanling pig coliform diarrhea that enterotoxigenic escherichia coli adhesin gene faeG is produced in the described expression of claim 1.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103233027A (en) * | 2013-04-24 | 2013-08-07 | 吉林农业大学 | Immune-enhanced escherichia coli ETEC (Enterotoxigenic E. Coli) resisting recombinant lactic acid bacteria and preparation method thereof |
| US20140086950A1 (en) * | 2012-09-24 | 2014-03-27 | Montana State University | Recombinant lactococcus lactis expressing escherichia coli colonization factor antigen i (cfa/i) fimbriae and their methods of use |
| CN107325998A (en) * | 2017-06-20 | 2017-11-07 | 江西嘉博生物工程有限公司 | A kind of Recombinant Lactococcus lactis and construction method for expressing pig's epidermal growth factor gene |
| CN107893111A (en) * | 2017-12-29 | 2018-04-10 | 武汉轻工大学 | A kind of screening technique of the feeding probiotics of anti-piglet epidemic diarrhea |
| CN112522172A (en) * | 2020-12-21 | 2021-03-19 | 中南民族大学 | Escherichia coli ETEC BE-311-faeG and application thereof |
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| 《中国预防兽医学报》 20090930 刘淑杰 等 ETEC粘附素蛋白亚基Fae G在乳酸乳球菌中的表达及免疫反应性分析 688-692 1-2 第31卷, 第9期 2 * |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20140086950A1 (en) * | 2012-09-24 | 2014-03-27 | Montana State University | Recombinant lactococcus lactis expressing escherichia coli colonization factor antigen i (cfa/i) fimbriae and their methods of use |
| US9452205B2 (en) * | 2012-09-24 | 2016-09-27 | Montana State University | Recombinant Lactococcus lactis expressing Escherichia coli colonization factor antigen I (CFA/I) fimbriae and their methods of use |
| US9931390B2 (en) | 2012-09-24 | 2018-04-03 | Montana State University | Recombinant Lactococcus lactis expressing Escherichia coli colonization factor antigen I (CFA/I) fimbriae and their methods of use |
| CN103233027A (en) * | 2013-04-24 | 2013-08-07 | 吉林农业大学 | Immune-enhanced escherichia coli ETEC (Enterotoxigenic E. Coli) resisting recombinant lactic acid bacteria and preparation method thereof |
| CN107325998A (en) * | 2017-06-20 | 2017-11-07 | 江西嘉博生物工程有限公司 | A kind of Recombinant Lactococcus lactis and construction method for expressing pig's epidermal growth factor gene |
| CN107893111A (en) * | 2017-12-29 | 2018-04-10 | 武汉轻工大学 | A kind of screening technique of the feeding probiotics of anti-piglet epidemic diarrhea |
| CN112522172A (en) * | 2020-12-21 | 2021-03-19 | 中南民族大学 | Escherichia coli ETEC BE-311-faeG and application thereof |
| CN112522172B (en) * | 2020-12-21 | 2022-05-17 | 中南民族大学 | Escherichia coli ETEC BE-311-faeG and application thereof |
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Application publication date: 20110427 |