CN101921725A - Converter for inhibiting hen somatostatin action through oral immunization and application thereof - Google Patents
Converter for inhibiting hen somatostatin action through oral immunization and application thereof Download PDFInfo
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- 238000002649 immunization Methods 0.000 title abstract description 6
- 230000003053 immunization Effects 0.000 title abstract description 5
- 229960000553 somatostatin Drugs 0.000 title description 6
- 102000005157 Somatostatin Human genes 0.000 title description 5
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Classifications
-
- Y02A50/472—
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a converter which comprises a recipient bacterium and a recombinant vector converted into the recipient bacterium. The recipient bacterium is lactococcus lactis NZ9800, an original carrier of the recombinant vector is pNZ8112, and exogenous target genes of the recombinant vector comprise a hen somatostatin gene, a cholera toxin B subunit gene and a cell wall anchor stator M6 gene. The converter of the invention can enter a hen intestine in an oral taking mode. Compared with injection immunization, the converter lowers the toxicity caused by directly contacting with a circulating system and enhances the vaccine safety. By selecting the combined immunization of an antigen gene and an immune enhancement factor, the invention enhances the body immune response and improves the immune effect.
Description
Technical field
The present invention relates to bioengineering field, relate in particular to the transformant and the application thereof of a kind of oral immunity blocking-up chicken Somatostatin effect.
Background technology
The indigenous chicken kind of China is compared with external kind, characteristics such as have the high degree of adaptability to surrounding environment, anti-low level management, disease resistance is strong, meat is good.But because China's indigenous chicken kind growth is slow, efficiency of feed utilization is low; a large amount of introducings along with external high productivity energy kind; cruel market competition makes the quantity of China's indigenous chicken kind in rapid decline; some kind even in imminent danger or be tending towards extinction, good and celebrated Xiaoshan chicken quantity significantly reduces with the big meat of build as element.Though external chicken kind growth is fast, efficiency of feed utilization is high, but meat flavor is poor, along with growth in the living standard, people more and more pay attention to the quality of meat, existing market is to the demand rapid growth of high-quality lean meat, this transformation to we proposed to cultivate that growth is fast, the requirement and the challenge of the good new variety of local flavor, strain.
Improve animal growth rate is that animal produces the target that the researchist made great efforts always.But improve breeding performonce fo animals by traditional genetic improvement and nutrition regulation means at present and be difficult to have again big progress.In order to improve the production performance of animal, in feed, add various forbidden drugs and cause the incident of food poisoning to happen occasionally, thereby to study a kind of thing growth method of actuating safe, efficient, that the mankind are had no side effect be the pressing issues that present aquaculture faces.
If immunologic method is applied to the regulation and control of breeding performonce fo animals, selection has inhibiting hormone or the factor and immunostimulant factor amalgamation and expression or common immunity to strengthen its immunogenicity to growth of animal, come immune animal as antigen, can bring out the create antagonism antibody of this kind hormone or the factor of body, thereby suppress or weaken the physiological function of this hormone or the factor.This technology is expected to improve the production performance of animal, and kind or the strain meaning slow to those growths are especially great, and fool proof without any side effects.
Because intestinal bacteria, yeast etc. can not field planting in animal intestinal, so can not carry out oral immunity, expressed purpose product purification must be carried out injecting immune.The protein purification step has increased production cost and difficulty greatly, makes scale operation target protein preparation be difficult to realize.Simultaneously the injecting immune program is also more loaded down with trivial details, not only will expend big manpower, to animal stress be also bigger with injury.Therefore press for develop a kind of to favorable safe, the convenient and oral preparations that can carry out scale operation of growth of animal.
Milk-acid bacteria is a class most widely used essential industry bacterial strain in food, and it comprises tens genus such as Lactococcus lactis, lactobacillus, is acknowledged as safe food-grade microorganisms.Milk-acid bacteria itself is exactly a kind of probiotic bacterium, can regulate intestinal microenvironment, helps digest, and suppresses the harmful intestinal tract microorganism, and control diarrhoea is actuated the thing growth-weight gain.
Summary of the invention
The invention provides the transformant of a kind of oral immunity blocking-up chicken Somatostatin effect, this transformant can be taken in also field planting in the chicken enteron aisle by oral way, its excretory fusion rotein can effectively bring out the antibody that produces anti-chicken Somatostatin in the chicken body, thereby suppresses or weaken the restraining effect of endogenous growth chalone to the chicken growth.
A kind of transformant, comprise recipient bacterium and the recombinant vectors that is transformed into recipient bacterium inside, described recipient bacterium is Lactococcus lactis NZ9800, the initial carrier of recombinant vectors is pNZ8112, and the external source goal gene of recombinant vectors comprises the sub-M6 gene of chicken somatostatin gene, b subunit of cholera toxin gene and cell walls grappling.The chicken Somatostatin (GenBank number: NM_205336) be a kind of 14 peptides, except that the hypothalamus secretion, organize also and can produce for Somatostatin, SST by gi tract and pancreas etc.SST not only has restraining effect to tethelin, can also suppress the secretion and the release of the gastrointestinal hormone ` that Regular Insulin, thyroxine and some promoting digestion absorb, thereby it is an important factor regulating growth of animal, and it is as immunogen in the present invention.
For the enhancing body immunne response, improve immune effect, on antigen gene, connect the b subunit of cholera toxin gene, b subunit of cholera toxin (Cholera toxin B subunit, be called for short CTB, GenBank number: U25679) be immunostimulant, studies show that it is a kind of avirulent very effective mucosa-immune adjuvant, is particularly suitable as the immunity enhancement adjuvant albumen of oral vaccine.
(GenBank number: M11338) can combine with the albumen on the Lactococcus lactis cytolemma, immunogen and immunostimulant are secreted into outside the born of the same parents after expressing the sub-M6 of cell walls grappling, and sub-M6 can be fixed on the cell walls by the cell walls grappling.
Initial carrier pNZ8112 contains the promotor NisA that strong promoter is nisin A (Nisin A) (GenBank number: AY303241) and efficient coding sequence of secretory signal peptide Usp45 (GenBank number: M60178), signal peptide Usp45 is positioned at the downstream of promotor NisA, and the external source goal gene is connected the C end of signal peptide.
The base sequence of described somatostatin gene, b subunit of cholera toxin gene, the sub-M6 gene of cell walls grappling, promotor NisA and signal peptide Usp45 is shown in SEQ ID NO.5～9.
Milk-acid bacteria itself is exactly a kind of probiotic bacterium, can regulate intestinal microenvironment, helps digest, and suppresses the harmful intestinal tract microorganism, and control diarrhoea is actuated the thing growth-weight gain.Milk-acid bacteria field planting or of short duration field planting in vivo is the prerequisite of its performance physiological function.By carrier goal gene such as immunogen gene are imported in the milk-acid bacteria, make active bacteria formulation then and feed, introduce in the animal body, thereby the product that produces the external source goal gene causes mucous membrane and humoral immunization.Find that after deliberation the lactobacillus of energy long period field planting makes animal that the purpose exogenous genes products is produced immunological tolerance easily in enteron aisle, and does not produce this immunological tolerance phenomenon at the short-and-medium tentative Lactococcus lactis of planting of enteron aisle.
The present invention also provides above-mentioned transformant in the active application of immunity blocking-up chicken Somatostatin, by with secreting, expressing the transformant feeding of external source goal gene product give chicken, Lactococcus lactis will of short duration field planting in the chicken enteron aisle, thereby cause that chicken produces immunity to external source goal gene product, promptly the Somatostatin that milk-acid bacteria is expressed produces immunity, produce the antibody of the long chalone of antibiosis, this antibody can weaken or suppress chicken endogenous growth chalone reaches the promotion growth to the restraining effect of chicken growth purpose.
According to above-mentioned application, the present invention provides a kind of chicken oral immunity preparation that utilizes above-mentioned transformant preparation again, and it comprises substratum and has secreted the transformant of external source goal gene product.Said preparation preparation and use are all very convenient.
Described substratum can be preferably the GM17 substratum for promoting the substratum of Lactococcus lactis growth.In use, transformant concentration is preferably 1 * 10 in the above-mentioned oral immunity preparation
8Cfu/mL～1 * 10
10Cfu/mL makes that the Lactococcus lactis in the enteron aisle reaches finite concentration, and each feeding amount is 1～2ml.
The present invention also provides a kind of preparation method of above-mentioned chicken oral immunity preparation, may further comprise the steps: transformant is placed substratum, be cultured to OD
600Be 0.3～0.5, add nisin and continue to cultivate 2～3h.
Described nisin is the induced expression thing, adds back concentration and is preferably 10～20ng/mL.
Transformant of the present invention can enter the chicken enteron aisle by oral way, compares with injecting immune, reduces directly to contact the toxicity that causes with the recycle system, increases the security of vaccine.Select antigen gene/immunostimulant factor (avirulent b subunit of cholera toxin) combined immunization for use, strengthened immune response, improved immune effect.
Embodiment
GM17 liquid nutrient medium: Tryptones 2%; Yeast extract paste 0.5%; Vc 0.05%; Extractum carnis 0.5%; Sodium acetate 0.15%; Sodium-chlor 0.4%; MgSO
4.7H
2O 0.25%; Glucose 0.5% transfers to pH 6.8.The GM17 solid medium adds 1.6% agar on above-mentioned medium base.
Lactococcus lactis NZ9800 and carrier pNZ8112 are commercially produced product, from the NIZO food research of Holland.
1, the structure of goal gene and clonal expression
(1) structure of CTB-SST mosaic gene
Upstream primer (SEQ ID NO.1):
5’-GG
ACACCTCAAAATATTACTG-3’
The primer structure is: protection base (GG)-Xba I site (sequence in the frame)-CTB 5 ' end parts sequence (underscore sequence)
Downstream primer (SEQ ID NO.2):
5’-TG
ACAGGATGTGAAAGTTTTCCAGAAGAAGTTCTTG CAGCCCGC 
-3’
Primer structure: protection base (TG)-BamH I site (sequence in the frame)-SST sequence (underscore)-linker (bold Italic sequence)-CTB 3 ' end parts sequence (double underline).
Is template with above-mentioned primer with cholera vibrio gene group DNA (containing the CTB gene), carries out PCR, and the PCR condition is: the total reaction system is 50 μ l, wherein contains the dna profiling of 50ng, 1 * PCR buffer, 1.5mM MgCl
2, 0.2mM dNTP, 0.2 μ M upstream and downstream primer, 2.5U Taq archaeal dna polymerase.The PCR reaction parameter is set to: 94 ℃ of pre-sex change 1 minute; 94 ℃ of sex change 30 seconds, 50 ℃ of annealing 30 seconds, 72 ℃ were extended 35 circulations 30 seconds; Extended 5 minutes after 72 ℃ then.
Because downstream primer self has SST, connexon linker and CTB 3 ' end parts sequence, therefore behind PCR, can directly obtain the CTB-SST mosaic gene.The PCR product is cut rear clone in pET28 carrier (Invitrogen company) with Xba I and BamH I enzyme.
The pET28 plasmid that will contain CTB-SST then is transformed in E.coli DH5 α (Takara company) competent cell, be coated with the LB flat board, cultivate 18-20h for 37 ℃, the positive spot enlarged culturing of picking, extracting plasmid then, PCR and enzyme are cut and are identified in the extractive plasmid and contain goal gene.
(2) structure of CTB-SST-M6 recombinant expression vector
Upstream primer (SEQ ID NO.3):
5’-AA
AGAGTGTTTCCTAGGGG-3’
The primer structure is: protection base (AA)-BamH I site (sequence in the frame)-M65 ' end parts sequence (underscore sequence)
Downstream primer (SEQ ID NO.4):
5’-CG
GTTTTCTTCTTTGCGTT-3’
The primer structure is: protection base (CG)-Hind III site (sequence in the frame)-terminator (bold Italic sequence)-M63 ' end parts sequence (underscore sequence)
With micrococcus scarlatinae genomic dna (containing the M6 gene) is template, carry out PCR (the PCR condition is the same) with above-mentioned primer, can obtain the PCR product of two ends band BamH I and Hind III restriction enzyme site, cut the PCR product with these two kinds of enzyme enzymes, be recombined into the CTB-SST gene downstream (the BamH I site by design links to each other CTB-SST with the M6 reorganization: (Xba I) CTB-linker-SST (BamH I) and (BamH I) M6-terminator (Hind III)) in the pET28 carrier that contains CTB-SST.
The pET28 plasmid that will contain the CTB-SST-M6 mosaic gene then is transformed in the E.coli DH5 α competent cell, be coated with the LB flat board, cultivate 18-20h for 37 ℃, the positive spot enlarged culturing of picking, extracting plasmid then, PCR and enzyme are cut and are identified in the extractive plasmid and contain goal gene.
The enzyme tangent condition is: 37 ℃ of enzymes are cut and are spent the night; Ligase enzyme and condition: T
4Dna ligase, 16 ℃ of connections are spent the night.Other processes are all carried out according to a conventional method.
2, the clone of CTB-SST-M6 mosaic gene in the intermediate host bacterium and the expression in Lactococcus lactis
(1) clone of CTB-SST-M6 mosaic gene in the intermediate host bacterium
Because the clone copy number of expression vector pNZ8112 (NIZO food research (Holland)) in Lactococcus lactis is very low, expect enough reorganization pNZ8112 plasmids (containing CTB-SST-M6), must make the pNZ8112 plasmid reach enough copy numbers by an intermediate host bacterium E.coli MCl061 (Invitrogen company).
Screen reorganization bacterium (10ug/ml paraxin) with chlorampenicol resistant: contain chloramphenicol resistance gene on the pNZ8112, E.coli MCl061 bacterial strain itself is not had a chlorampenicol resistant, and the E.coli MCl061 that only is transformed into the pNZ8112 plasmid could grow containing on the flat board of paraxin.
At first goal gene (CTB-SST-M6) enzyme from the pET28 plasmid is cut (Xba I and HindIII) and get off, rubber tapping is reclaimed, and is recombined among the pNZ8112 that cut with same enzyme T
416 ℃ of connections of dna ligase are spent the night, and Transformed E .coli MC1061 competent cell is coated with the LB flat board then, cultivate 18-24h for 37 ℃, the bacterial plaque enlarged culturing that grows on the picking paraxin flat board, extracting plasmid then, PCR and enzyme are cut and are identified in the extractive plasmid and contain goal gene.
(2) expression of CTB-SST-M6 mosaic gene in Lactococcus lactis
After obtaining containing the reorganization E.coli MC1061 of goal gene (CTB-SST-M6), with its enlarged culturing, make reorganization pNZ8112 plasmid wherein obtain enough copy numbers (about 10～60 parts of copies of each host cell), extracting reorganization pNZ8112 plasmid then, be transformed in Lactococcus lactis NZ9800 (NIZO food research (the Holland)) competent cell, coating contains the GM17 flat board (equally with chlorampenicol resistant screening reorganization bacterium) of 10ug/ml paraxin, cultivate 48-60h in 30 ℃, the bacterial plaque enlarged culturing that picking grows, extracting plasmid, PCR and enzyme are cut in the extractive plasmid of evaluation and are contained goal gene.
The Lactococcus lactis that will contain positive recombinant plasmid is transferred to 5ml and contains in the GM17 liquid nutrient medium of paraxin (final concentration is 10ug/ml), behind 30 ℃, 180r/min overnight incubation; To the 10ml fresh culture, 30 ℃ were cultured to OD600 and are about 0.4 by 1: 25 dilution proportion, and adding final concentration then is that 10ng/ml nisin nisin (Sigma company) induces destination gene expression 2-3h, and bacterium liquid is stand-by.
(3) check target protein
At first the thalline in the above-mentioned bacterium liquid is handled with N,O-Diacetylmuramidase, use ultrasonic disruption again, cell solution that then will be after ultrasonic disruption is handled with 70% saturated ammonium sulphate after, the centrifugal 30min of 10000g, with resolution of precipitate in 50mMol/L pH6.0 phosphoric acid buffer, dialyse with the phosphoric acid buffer of 50mMol/L behind the activated carbon decolorizing and detect to the dialyzate less than the ammonium radical ion, directly go up DEAE-Cellulose 52 post (2cm * 60cm, Pharmacia, in advance with the pH6.0 that contains 30mMol/L NaCl, the phosphoric acid buffer balance of 20mMol/L), carry out linear gradient elution with buffer system (the 20mMol/L pH6.0 phosphoric acid buffer and the 20mMol/L pH6.0 phosphoric acid buffer that contains 300mMol/L NaCl that contain 30mMol/L NaCl), after washing post with initial damping fluid (the 20mMol/L pH6.0 phosphoric acid buffer that contains 30mMol/L NaCl) earlier, carrying out NaCl concentration again is the linear gradient wash-out of 30-300mMol/L, elution flow rate is 12mL/h, collect target protein with nucleic acid/Protein Detection instrument monitoring, use the deionized water dialysed overnight, again after the freeze concentration in 4 ℃ of preservations, with conventional Western blotting (Western blotting) detection validation, detection method is as follows:
At first use commercial SST antigen (Sigma product) immune rabbit, it is one anti-that preparation is used for that Western blotting detects, and is two to resist with the goat anti-rabbit igg antibody (Sigma product) of commercial horseradish peroxidase-labeled.For whether testing goal PROTEIN C TB-SST-M6 (the gene size is 1674bp) is expressed by Lactococcus lactis, we use the expression product and another kind of known SST fusion rotein (the Salmonellas flagellin encoding gene (H-1 of purifying
d)-Somatostatin is called for short H-1
d-SST, gene size is 1542bp) compare, if with prepared one the resisting and can combine of commodity SST antigen immune rabbit with purified product, again can with H-1
d-SST protein binding, and the molecular weight of albumen size conforms to then proves in the expression product of purifying to contain target protein CTB-SST-M6.Western blotting result shows: H-1
d-SST and purified product all can close with the prepared resistive connection of commodity SST antigen immune rabbit, demonstrate pairing protein band (H-1
d-SST molecular weight is 40～50KD, and CTB-SST-M6 is 50～60KD), illustrates that the CTB-SST-M6 fusion rotein is by the Lactococcus lactis successful expression.
3, the antibody production behind the CTB-SST Lactococcus lactis oral immunity chicken
40 AA chickens of picked at random have carried out the recombinant lactic acid bacteria oral immunity, are 1 * 10 with concentration
9The bacterium liquid feeding of cfu/mL is the chick (1 age in days) of birth just, each feeding 1～2ml, 2 times weekly, 3 weeks of feeding.Certainly change bacterial concentration, generally select 1 * 10 according to practical situation
8～1 * 10
10Cfu/mL.
3 weeks, 6 weeks and 8 weeks are extracted chicken blood after first immunisation, separate obtaining chicken serum, are used to detect chicken SST antibody production.Adopt the antibody horizontal in the ELISA indirect method mensuration chicken serum, concrete steps are as follows:
SST antigen (Sigma product) with 0.5mg/ml wraps by 96 holes immunity plate, every hole adds antigenic solution 100ml, 48 ℃ of bags are spent the night, then with containing 1.5%BSA (bovine serum albumin, the Sigma product) PBST solution (the PBS solution that contains 0.05%Tween-20) seals immune plate, 37 ℃ of sealing 2h.The adding extent of dilution is 1: 50 a chicken serum to be measured, wash three times, adding extent of dilution again is the goat-anti chicken igg antibody (Sigma product) of 1: 1000 horseradish peroxidase-labeled, then with 10mg 3,3 ', 5,5 '-tetramethyl benzidine (Sigma product, the substrate of enzyme can show blue under the horseradish peroxidase effect) be dissolved in the 0.025M phosphoric acid salt citrate buffer solution and add in each hole of immune plate every hole 50ml.Add 0.2M H after the substrate colour developing
2SO
4Termination reaction, absorbance value (OD value) after developing the color with wavelength 450nm mensuration substrate with enzyme connection determinator then, to record sample (being the immune chicken serum sample) OD value (P) compares with negative control (not immune chicken serum sample) OD value (N), if the P/N value is more than or equal to 2.0, then institute's this antibody of test sample is positive, and chicken SST antibody production detected result is as shown in the table:
4, CTB-SST viable lactic acid bacteria preparation active immunity is to the influence of chicken production performance
Chosen 20 of antibody positive as immune group from above-mentioned 40 AA chickens of immunity, select 20 not immune chickens else and organize in contrast.The AA chicken is weighed when raising to 56 ages in days after the above-mentioned bacterium liquid active immunity, adds up day weight gain and the material anharmonic ratio of chicken simultaneously, and the result is as shown in the table:
Immunological reagent active immunity of the present invention is to AA growth of meat chicken Effect on Performance
Different subscript letter representation significant differences in the same column, a, b represent p＜0.05, a, c represents p＜0.01.
Above data show that the chicken CTB-SST viable lactic acid bacteria preparation that the present invention developed can stimulate chicken to produce the antibody of the long chalone of antibiosis (SST), weakens the restraining effect of body somatostatin to the chicken growth, has improved the speed of growth and the feed conversion rate of chicken.
Claims (7)
1. transformant, comprise recipient bacterium and the recombinant vectors that is transformed into recipient bacterium inside, it is characterized in that: described recipient bacterium is Lactococcus lactis NZ9800, the initial carrier of recombinant vectors is pNZ8112, and the external source goal gene of recombinant vectors comprises the sub-M6 gene of chicken somatostatin gene, b subunit of cholera toxin gene and cell walls grappling.
2. the described transformant of claim 1 is in the active application of immunity blocking-up chicken endogenous growth chalone.
3. a chicken oral immunity preparation that utilizes the described transformant preparation of claim 1 is characterized in that: comprise substratum and the transformant of having secreted described external source goal gene product.
4. chicken oral immunity preparation according to claim 3 is characterized in that: described substratum is the GM17 substratum.
5. chicken oral immunity preparation according to claim 3 is characterized in that: described transformant concentration is 1 * 10
8Cfu/mL～1 * 10
10Cfu/mL.
6. the preparation method of chicken oral immunity preparation according to claim 3 may further comprise the steps: transformant is placed substratum, be cultured to OD
600Be 0.3～0.5, add nisin and continue to cultivate 2～3h.
7. according to the preparation method of claim 6, it is characterized in that: it is 10～20ng/mL that described nisin adds back concentration.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996011277A1 (en) * | 1994-10-05 | 1996-04-18 | Dompe' S.P.A. | Microorganisms as therapeutic delivery systems |
| CN101168741A (en) * | 2007-09-28 | 2008-04-30 | 中国疾病预防控制中心传染病预防控制所 | Lactococcus lactis food-sate secretion expression carrier and its preparing method and application |
| EP0871748B1 (en) * | 1995-10-20 | 2009-07-01 | Actogenix N.V. | Delivery of biologically active polypeptides |
-
2010
- 2010-07-27 CN CN2010102380863A patent/CN101921725B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996011277A1 (en) * | 1994-10-05 | 1996-04-18 | Dompe' S.P.A. | Microorganisms as therapeutic delivery systems |
| EP0871748B1 (en) * | 1995-10-20 | 2009-07-01 | Actogenix N.V. | Delivery of biologically active polypeptides |
| CN101168741A (en) * | 2007-09-28 | 2008-04-30 | 中国疾病预防控制中心传染病预防控制所 | Lactococcus lactis food-sate secretion expression carrier and its preparing method and application |
Non-Patent Citations (3)
| Title |
|---|
| 《Biotechnology of Lactic Acid Bacteria Novel Applications》 20100325 Anderson Miyoshi et al Lactic Acid Bacteria as Live Vectors Heterologous Protein Production and Delivery Systems , 1 * |
| 《Camp. Biochem. fhysiol》 19861231 G.S.G.SPENCER et al The effect of immunization against somatostatin on growth rates and growth hormone secretion in the chicken 第85A卷, 第3期 2 * |
| 《Folia Microbiol》 19951231 P.M. NORTON et al Progress in the development of Lactococcus lactis as a recombinant mucosal vaccine delivery system 第40卷, 第3期 2 * |
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