CN1195062C - Non-antibiotics resistant shuttle plasmid expression carrier and its constructing method and uses - Google Patents
Non-antibiotics resistant shuttle plasmid expression carrier and its constructing method and uses Download PDFInfo
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- CN1195062C CN1195062C CNB031004156A CN03100415A CN1195062C CN 1195062 C CN1195062 C CN 1195062C CN B031004156 A CNB031004156 A CN B031004156A CN 03100415 A CN03100415 A CN 03100415A CN 1195062 C CN1195062 C CN 1195062C
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Abstract
The present invention relates to a non-antibiotic resistant shuttle plasmid expressing carrier, a construction method thereof and an application thereof, which belongs to the technical field of biology engineering. A basic plasmid carrier pMG36e forms a line type by carrying out enzyme cut on an enzyme cutting site of restriction endonuclease EcoRI by the restriction endonuclease EcoRI, an end near to erythrocin Er is connected with a non-antibiotic resistant gene and then is cyclized back to a ring shape, and then the erythrocin gene Er is removed by endonuclease on the ring to form the non-antibiotic resistant shuttle plasmid expressing carrier. Protective antigen genes of human or animal diseases are connected to the non-antibiotic resistant shuttle plasmid carrier of the present invention to manufacture functional foods, bacterial suspensions with a preventing and protecting function to specific diseases, oral tonic prescriptions, tube feed products and animal vaccines. The present invention uses a non-antibiotic feasible gene for replacing an erythrocin gene and does not have the problem of the diffusion of antibiotic resistant genes in the process of use.
Description
Technical field
The invention belongs to technical field of bioengineering, relating to non-antibiotic resistance shuttle plasmid expression vector reaches with the antibiotic resistance gene being the non-antibiotic gene Selection pressure remodeling method of selective pressure plasmid vector, adopting said method makes up the plasmid expression vector of non-antibiotic gene Selection pressure, and this carrier is applied in the Lctobacteriaceae microorganism, particularly be applied in the lactobacillus microorganism belonging to genus.Use in the lactobacillus microorganism belonging to genus by this carrier and its, can be used for preventing, treating specific eqpidemic disease or given play to growth regulating function and the trophic function that it has.Particularly, the present invention relates to the antibiotic resistance gene being the non-antibiotic gene Selection pressure modification scheme of selective pressure plasmid vector and with the plasmid vector of this scheme constructs and use the upholder of ingesting of non-antibiotic gene Selection pressure plasmid vector preparation and contain the composition of this upholder.
Technical background
Behind the milk-acid bacteria of pasteur (Pasteur) discovery fermentation yogurt in 1857, there is the people to be separated to bifidus bacillus and Lactobacillus acidophilus from enteron aisle in succession again, from then on opened up new era of milk-acid bacteria research.
Milk-acid bacteria is a main member in the biosphere, they are distributed widely in nature, in soil, water, plant, especially are present in people and animals' digestive tube, except that minority, the overwhelming majority is a requisite normal microflora with important physiological function in the humans and animals body.Milk-acid bacteria is as one of the important member of normal microflora in active bacteria formulation and the humans and animals body, and is closely bound up with the life of the mankind and animal, and its application in life science is subjected to people's attention day by day and obtains the extensive exploitation utilization.
Because the F-strain of reorganization protective antigen mainly is the attenuation pathogenic bacteria that lives, and the immune protective efficiency of bacterial vaccine often is directly proportional with the remaining virulence of bacterial strain.Be subjected to people's attention day by day so attempt to seek the F-strain that to express exogenous antigen and safety well.The important member of normal microflora in lactobacillus behaviour and the animal body; with the F-strain of lactobacillus as expression external source protective antigen gene; just the biological function of milk-acid bacteria and the specific immunity of external source function antigen gene can be combined; simultaneously lactobacillus is as the normal physiological bacterium, has the characteristics such as oral safe, convenient, cheapness of new generation vaccine again.Succeeding in developing of this class vaccine will be explored a new road for the development of new generation vaccine.
But Bao Dao humans and animals digestive tube symbiosis lactobacillus expression vector system all is with pressure and the selection markers of antibiotics resistance as the foreign gene stably express up to now.After the exogenous antigen gene is inserted into the polyclone restriction enzyme site, plasmid vector on the selection flat board that adds the antibiotics resistance selectable marker owing to do not insert the plasmid vector of foreign gene and can not survive, and the plasmid vector that inserts foreign gene can be grown, so be easy to screen the positive colony plasmid vector, but along with antibiotic widespread use or even abuse, antibiotic resistance gene diffusion problem also is on the rise, thereby this class expression system also has very big distance from the requirement of " edibility ", can not be directly used in humans and animals.Because constantly drift diffusion in environment of drug resistance gene may cause the destruction to the little ecology of environment, brings the serious consequence that can't imagine, some bacterium almost all produces resistance-occurred superbacteria to all microbiotic now; And drug resistance gene can transmit between bacterium by plasmid and cause drug resistance gene big area diffusion, popularize, and no matter to human body microenvironment, animal internal milieu, still the Nature ecotope all caused severe contamination.According to World Health Organization's statistics, it is the superinfection that causes antibiotic medicine to lose efficacy and cause owing to the drug resistance gene diffusion that there is the cause of the death of patient in a lot of hospitals in the annual whole world.Non-antibiotic selective pressure plamid vector construction strategy can be avoided a series of problems of causing owing to the transmission of antibiotics resistance gene between plasmid vector.So making up with the non-antibiotic resistance is that the lactobacillus expression system of " food grade " and " feed grade " of mark and selective pressure just seems very necessary.And have huge use value and a market application foreground.
Summary of the invention
The present invention causes drug resistance gene big area diffusion, universal in order to overcome because drug resistance gene transmits between bacterium by plasmid, to human body microenvironment, animal internal milieu, the Nature ecotope causes the severe contamination problem, by making up with the non-antibiotic resistance is the lactobacillus expression system of " food grade " and " feed grade " of mark and selective pressure, purpose provides a kind of non-antibiotic resistance shuttle plasmid expression vector and construction process and application, fundamentally solves the problem of drug resistance gene diffusion.
Non-antibiotic resistance of the present invention is the selective pressure of one of essential characteristic at plasmid vector, because used plasmid vector all is to be selective pressure with the antibiotics resistance gene at present, as erythromycin gene, kanamycin gene, chloromycetin gene etc., be to belong to antibiotics resistance selective marker expression vector, owing to have the harm of genetic drift diffusion, make the antibiotics resistance plasmid expression vector transform the harm that non-antibiotic r plasmid expression vector just can be stopped the antibiotics resistance gene diffusion as if the antibiotics resistance gene screening mark in the plasmid expression vector is replaced with other non-antibiotic gene.The present invention is exactly according to this method, substitute antibiotic resistance gene in the original plasmid vector as selective pressure or selection markers with non-antibiotic resistant gene NASG (NASG is a Non-antibioticSelective Genes english abbreviation), (gene of β-Gal), plasmid vector has just fundamentally been avoided the harm of antibiotics resistance gene diffusion in to the animals and plants application process like this for non-antibiotic resistant gene NASG such as thymidylate synthetase (ThyA) gene or beta-galactosidase enzymes.Of the present invention shuttling back and forth is meant that above-mentioned plasmid expression vector both can (remove from office blue Albert'stain Albert for blue at leather Lan Shi positive bacteria, as, milk-acid bacteria) expresses in, can in the cloudy row of leather Lan Shi bacterium (remove from office blue Albert'stain Albert, as, intestinal bacteria), express again for red, and this kind plasmid carrier that extracts from negative bacterium can enter and duplicates expression in the positive bacteria, vice versa, passes through between two bacterioids, expresses, so be called the shuttle expression plasmid carrier as shuttle.
Non-antibiotic resistance shuttle expression plasmid carrier of the present invention is basic plasmid pMG36e, cuts through restriction enzyme EcoR I enzyme on its restriction enzyme site, makes it become line style.Connect non-antibiotic resistant gene, cyclisation winding shape afterwards at nearly Erythromycin E r end.On this ring, erythromycin gene Er is removed with restriction endonuclease again, form non-antibiotic resistance shuttle expression plasmid carrier, represent with pMG425t.
Non-antibiotic resistance shuttle expression plasmid carrier construction method of the present invention has versatility, and promptly any non-antibiotic resistant gene NASG that can be used as the plasmid expression vector selection markers all can be applied in this construction step and reach identical purpose.Be the non-antibiotic resistant gene as long as its two ends have or the restriction enzyme site PstI and the NsiI that add restriction enzyme can be connected in the original vector, be configured to a new non-antibiotic resistance expression carrier.
Non-antibiotic resistance shuttle expression plasmid carrier of the present invention is characterized in that (microbiotic (erythromycin) gene among the original plasmid vector pMG26e of gene substitution of β-Gal) is as selective pressure or selection markers with thymidylate synthetase (ThyA) gene or beta-galactosidase enzymes, can (remove from office blue Albert'stain Albert at leather Lan Shi positive bacteria for blue, as, milk-acid bacteria) and the cloudy row of leather Lan Shi bacterium (remove from office blue Albert'stain Albert for red, as, intestinal bacteria) shuttling expressing between, become plasmid vector pMG425t, as shown in Figure 7.
Non-antibiotic resistance shuttle expression plasmid carrier pMG425t of the present invention has following characteristics: selectable marker gene is the non-antibiotic resistant gene, as thymidylate synthase gene (ThyA), beta-galactosidase gene (β-Gal), rather than antibiotics resistance gene; It can promptly be removed from office in Lan Shi negative bacterium and the leather Lan Shi positive bacteria and carry out shuttling expressing at intestinal bacteria and lactobacillus; Carrier can have complementary functions in the intestinal bacteria of ThyA or β-Gal gene defection type and lactobacillus and make the F-strain healthy growth.
The construction process of non-antibiotic resistance shuttle vectors of the present invention is basic plasmid pMG36e, cuts through restriction enzyme EcoR I enzyme on its restriction enzyme site, becomes line style.Connect non-antibiotic resistant gene, cyclisation winding shape afterwards at nearly Erythromycin E r end.On this ring, erythromycin gene Er is removed with restriction endonuclease again, form non-antibiotic resistance shuttle expression plasmid carrier.
The structure of non-antibiotic resistance shuttle vectors of the present invention is to be basic plasmid with pMG36e.PMG36e is common shuttling back and forth of the milk-acid bacteria of publishing, can be in intestinal bacteria and milk-acid bacteria shuttling expressing, size is 3611bps, erythromycin resistance gene is a selection markers, P-32 is a promotor, a part of restriction endonuclease sites such as Fig. 1 in the carrier.
Restriction enzyme kind and position according to the antibiotics resistance gene both sides, the non-resistance selection markers candidate gene of design primer amplification, as thymidylic acid synthase gene ThyA or beta-galactosidase gene (β-gal), be connected in the carrier that contains antibiotics resistance gene, thoroughly remove antibiotics resistance gene with restriction enzyme at last.Here the non-antibiotic resistant gene just is equivalent to the effect of antibiotics resistance screening-gene.
Restriction endonuclease is meant the general name of retriction endonuclease, is used for cutting, connects, and is meant EcoR I here, and Nsi I, and the restriction enzyme site restriction endonuclease in the accompanying drawing 1,6 are as Nde I, Mun I, Ecoo109 I, NheI, Hpa I, Bc II, Sac I, Ban II, Bsp 12861, Xma I, Sma I, Cla I, XbaI, Sal I, Acc I, Pst I, Sph I, Hind III, Eae I, Ban I, Acc 651, KpnI, Eco47 III, BamH I, EcoR II, Hinc II, Sac II.
Because the non-antibiotic resistant gene itself varies in size, for example the ThyA gene has 1132 bases (bp), and β-Gal gene has 3660 bases (bp), so be connected to carrier varying in size of novel vector later on; The used F-strain of carrier construction expression system is also different; Comparatively speaking, the external source fragment charge capacity of carrier that with the ThyA gene is selective pressure is big, and be that the external source fragment charge capacity of carrier of selective pressure is little with β-Gal gene, just β-Gal gene itself has the function of secreting signal peptide, so it should more help expressing less relatively external source fragment.But with ThyA gene and β-Gal gene is that the construction process of the plasmid vector of selective pressure is consistent.Be that the construction process of selective pressure plasmid vector describes only below in order to the ThyA gene.
The construction process detailed process of non-antibiotic resistance shuttle vector of the present invention now is described in detail in detail.
(1) with restriction endonuclease EcoR I plasmid vector pMG36e is carried out enzyme and cut, make cyclic DNA become line style, handle the plasmid vector that is cut to line style by EcoR I enzyme with polysaccharase T4 DNA again, be connected with the non-antibiotic resistant gene in order to next step.
(2) amplification non-antibiotic resistant gene ThyA.Chromosomal DNA with lactobacillus johnsonii is a template, and the lactobacillus johnsonii ThyA gene order according to having delivered has designed a pair of primer, holds at 5 ' of primer to be added with PstI and NsiI restriction enzyme site respectively.The expression formula of designed primer is as follows:
Primer1:5 ' TC
CTGCAG3 ' 25 bases of CAC AGC TTG ATG CGA TC
PstI
Primer2:5 ' TT
ATGCAT3 ' 26 bases of GTG TCA TTG GTA AAC CTG
NsiI carries out polymerase chain reaction PCR amplification ThyA gene with high-fidelity polysaccharase Tag PlusI.
(3) the non-antibiotic resistant gene ThyA that obtains in the basic plasmid pMG36e that has been cut to line style in (1) and (2) is connected, transforms, be the ThyA gene and being connected and conversion of plasmid vector pMG36e, wherein in connection procedure, used linear plasmid carrier pMG36e and the ratio that is connected of non-antibiotic resistant gene ThyA are between 1: 1～1: 10.This moment, carrier had two resistances, i.e. the plasmid vector pMG425et of erythromycin gene and ThyA gene.
Because being connected with non-antibiotic resistant gene ThyA in the conversion process to exist at the basic plasmid pMG36e of line style connects and the problem of closure mistake, so must determine exactness and the directivity that the ThyA gene connects.For exactness and the directivity that determine to connect, with above-mentioned be the bacterial strain of selective pressure with two resistances (erythromycin and thymidine or beta-galactosidase enzymes), carry out the little extracting of plasmid, electrophoresis.Select positive strain wherein to carry out the big extracting of plasmid according to electrophoresis result, carry out enzyme with restriction endonuclease NsiI again and cut evaluation, with exactness and the directivity of determining that the ThyA gene inserts.
(4) thoroughly remove erythromycin resistance gene among the plasmid vector pMG425et.With restriction endonuclease EcoR I and Nsi I the plasmid vector pMG425et that has two resistances and comprise erythromycin gene and ThyA gene is carried out enzyme and cut, purpose is to be thoroughly to remove erythromycin resistance gene.Thoroughly cut in order further to identify that erythromycin gene among the plasmid vector pMG425et determines whether from the molecular weight angle, the extraction plasmid of positive bacteria is carried out enzyme with restriction endonuclease Hinc II cut evaluation.
In order to determine whether erythromycin gene is thoroughly removed, must carry out the erythromycin resistance screening identifies, identify recombinant vectors containing on the LB substratum of erythromycin, whether cut with definite confirmation erythromycin gene, because if thoroughly excised, then this bacterium just can not grow containing on the LB substratum of erythromycin.Otherwise, then can grow.
For the existence of determining the ThyA gene among the plasmid vector pMG425t also will be carried out the evaluation of the repairing effect of plasmid vector pMG425t, the intestinal bacteria that are the ThyA genetic flaw can not grow or poor growth on common LB substratum, must be added with could recover under the thymidine condition of external source the growth.Contain the intestinal bacteria that changing over to of ThyA gene recombination plasmid can make the ThyA genetic flaw and on basic medium, recover growth, the bacterial strain that contains recombinant vectors pMG425t that obtains is continued to go down to posterity not containing on the LB substratum of thymidine or beta-galactosidase enzymes, positive colony is extracted plasmid, as Fig. 6.
Non-antibiotic resistance shuttle vectors of the present invention can be applicable to manufacturing function dairy products and fodder additives and animal vaccine.
The protective antigen gene of human or animal's disease is connected into non-antibiotic r plasmid carrier of the present invention; again the carrier electricity is swashed and be transformed in the lactobacillus F-strain; promptly can manufacturing function food with this transgenosis lactobacillus, as function milk, sour milk, curdled milk, fermented milk, milk based powders, the milk base leavened prod of anti-specified disease.
In addition; the protection of animal antigen gene is connected into non-antibiotic r plasmid carrier of the present invention; again carrier electricity is swashed and be transformed in the lactobacillus F-strain, specified disease is had the bacterial suspension of prevention, provide protection, dried oral tonic, wet oral tonic, dried tube feed product or wet tube feed product, animality vaccine and have the additive of growth regulating function and trophic function with this transgenosis lactobacillus manufacturing.
The construction process of non-antibiotic resistance shuttle vectors of the present invention is used the non-antibiotic feasible base because of ThyA/ β-gal gene substitution erythromycin gene, the problem that does not in use have the antibiotics resistance gene diffusion, and then given prominence to environmental consciousness and effect, this all has great importance in theory and practice.
Description of drawings
Fig. 1 is used basic plasmid pMG36e structure iron and a part of restriction endonuclease sites of the present invention, Ori is the replication initiation gene among the figure, Er is an erythromycin resistance gene, P32 is phosphorus 32 promoter genes, MCS is a multiple clone site, T is the terminator gene, and EcoR I 2476, Ec47 III 3319 etc. reach the position of relative 1/0 deoxyribonucleotide in carrier for the restriction enzyme title.
Fig. 2 is the pcr amplification product electrophoresis image of the used non-antibiotic resistance ThyA gene of the present invention, 1 is λ DNA/EcoR I and Hind III standard DNA molecular weight among the figure, 2,3,5 is the ThyA gene of pcr amplification, and size is 1132bp, 4 negative results of comparison.
Fig. 3 cuts evaluation electrophoresis image for the enzyme that linear plasmid carrier pMG36e and ThyA gene oppositely are connected back recombinant vectors pMG36e+ThyA, and M is a λ DNA/EcoRI+HindIII standard molecular weight among the figure, and 75 cut the result with Hinc II enzyme for carrier pMG425t.
Fig. 4 cuts evaluation electrophoresis image for linear plasmid carrier pMG36e is connected afterwards the enzyme of recombinant vectors pMG425t+ThyA with ThyA gene forward, and M is a λ DNA/EcoRI+Hind III standard molecular weight among the figure, and 36 cut the result with Hinc II enzyme for carrier pW425t.
Fig. 5 identifies that for cutting with restriction enzyme Hinc II enzyme recombinant plasmid vector pW425et determines whether thorough removed enzyme is cut evaluation electrophoresis image to erythromycin gene, 1 is λ DNA/EcoR I+HindIII standard molecular weight among the figure, and 2,3 cut the result with Hinc II enzyme for recombinant vectors pMG425t.
Fig. 6 for of the present invention be the lactic acid bacteria shuttle expression plasmid carrier physics structural representation of selective pressure with non-antibiotic resistance ThyA gene, Ori is the replication initiation gene among the figure, ThyA is the thymidylic acid gene, P32 is phosphorus 32 promoter genes, MCS is a multiple clone site, T is the terminator gene, and EcoR I2021, Ec47 III 3424 etc. reach the position of relative 1/0 deoxyribonucleotide in carrier for the restriction enzyme title.
Fig. 7 is an operational flowchart of the present invention.Wherein pMG36e is a basic plasmid, it contains erythromycin resistance gene, can in intestinal bacteria and lactobacillus, express, NASG is non-antibiotic resistant gene (Non-Antibiotic Selective Genes), Er represents erythromycin resistance gene, and+number expression connects, and-number expression is removed,=number expression forms, and pMG425t represents non-antibiotic resistance shuttle expression plasmid carrier of the present invention.
Fig. 8 is a schema of operation of the present invention.Wherein pMG36e is a basic plasmid, it contains erythromycin resistance gene, can in intestinal bacteria and lactobacillus, express, ThyA is a thymidylate synthase gene, Er represents erythromycin resistance gene, and+number expression connects, and-number expression is removed,=number expression forms, and pMG425t represents non-antibiotic resistance shuttle expression plasmid carrier of the present invention.
Fig. 9 is schema of operation of the present invention.Wherein pMG36e is a basic plasmid, it contains erythromycin resistance gene, can in intestinal bacteria and lactobacillus, express, (β-Gal) is a beta-galactosidase gene, Er represents erythromycin resistance gene, and+number expression connects, and-number expression is removed,=number expression forms, and pMG425t represents non-antibiotic resistance shuttle expression plasmid carrier of the present invention.
Embodiment
Restriction enzyme kind and position according to the antibiotics resistance gene both sides, the non-resistance selection markers candidate gene of design primer amplification, at first basic plasmid pMG36e being carried out enzyme with restriction endonuclease EcoR I at 2476 cuts, make ring-like plasmid vector become line style, with polysaccharase T4 DNA the pMG36e plasmid vector and the ThyA gene of line style are handled, add the A/T tail at otch, again the ThyA gene is connected to cyclisation in the line style carrier that contains antibiotics resistance gene, thoroughly removes antibiotics resistance gene with restriction enzyme Nsi I and EcoR I at last.
(1) plasmid vector pMG36e is carried out enzyme with restriction endonuclease EcoR I at 2476 and cut to make and become line style, handle the plasmid vector that EcoR I enzyme is cut to line style with the T4 archaeal dna polymerase again, be beneficial to next step and be connected with the external source fragment.
(2) amplification non-antibiotic resistance screening marker gene ThyA.Chromosomal DNA with lactobacillus johnsonii is a template, and the sequence according to the lactobacillus johnsonii ThyA gene of having delivered has designed a pair of primer, holds at 5 ' of primer to be added with PstI and NsiI restriction enzyme site respectively.Carry out pcr amplification ThyA gene with high-fidelity polysaccharase Tag Plus I.
Primer design:
Primer1:5 ' TC
CTGCAG3 ' 25 bases of CAC AGC TTG ATG CGA TC
PstI
Primer2:5 ' TT
ATGCAT3 ' 26 bases of GTG TCA TTG GTA AAC CTG
NsiI
Reaction system: add each composition by following formula in the 500 microlitres sterilization PCR pipe:
10 times of PCR damping fluid 5.0 microlitres
10 mmole d NTP, 2.5 microlitres
100 picomole Primer, 1 0.5 microlitres
100 picomole Primer, 2 0.5 microlitres
L.caseiDNA template 2.0 microlitres
D.D.W. 38.5 microlitres
The 5 Taq PlusI of unit archaeal dna polymerases, 1.0 microlitres
Cumulative volume 50.0 microlitres
Mixing, instantaneous centrifugal after, add 50 microlitres (μ L) sterilising liq paraffin again and cover, carry out 94 ℃ of sex change 8 minutes on the PCR instrument, carry out following reaction again, 94 ℃ of sex change 1 minute, 51 ℃ of annealing 1 minute, 72 ℃ were extended 30 circulations 2 minutes.72 ℃ were extended 10 minutes.Reaction finishes the back and carries out purifying recovery ThyA gene with test kit, as Fig. 2.
(3) the carrier pMG36e that will add the A/T tail is connected with non-antibiotic resistant gene ThyA gene, be non-antibiotic resistant gene ThyA gene 6 microlitres, 2 microlitre line style pMG36e, T4 dna ligase 2 microlitres, 10 times of T4 dna ligase damping fluid 2 microlitres, distilled water 8 microlitres.At first 14 ℃ of connections are spent the night, and 4 ℃ of connections are spent the night again, and here linear plasmid carrier pMG36e is 1: 3 with the ratio that is connected of non-antibiotic resistant gene.The conversion of ThyA gene and vector plasmid pMG36e in 1.5 milliliters of centrifuge tubes of precooling, adds 200 microlitre competent escherichia coli cell DH5 α, connector 20 microlitres, mixing gently, ice bath 40 minutes, 42 ℃ of water-bath heat shocks 2 minutes.Add and to be preheated to 37 ℃ LB liquid nutrient medium 800 microlitres, 37 ℃, 200 rpms joltings 1 hour are coated the LB culture medium flat plate that is added with erythromycin, 37 ℃ of incubator overnight incubation.Picking LB (be added with erythromycin, do not add exogenous thymidine) goes up a youngster clone at random, extracts plasmid and evaluation after the enlarged culturing.This moment, carrier was for having the plasmid vector pMG425et of two resistance-erythromycin genes and ThyA gene.
The LB substratum comprises peptone (bacto-tryptone) 10 grams, yeast extract (bacto-yeastextract) 5 grams, NaCl 10 grams.
(4) determine exactness and the directivity that the ThyA gene inserts.The above-mentioned pair resistances (erythromycin resistance gene and thymidylic acid synthase gene) that contain are the bacterial strain of selective pressure plasmid vector pMG425et, carry out the little extracting of plasmid, electrophoresis.Select positive strain wherein to carry out big extracting plasmid according to electrophoresis result, carry out enzyme with the NsiI enzyme and cut evaluation, with exactness and the directivity of determining that the ThyA gene inserts.If the ThyA gene is reverse connection, should produce 2584 bases and 2159 base two bar segment after cutting with Nsi I enzyme.Connect if the ThyA gene is a forward, should produce 3716 bases and 1027 base two bar segment after cutting with Nsi I enzyme, as Fig. 3,4.
(5) erythromycin gene in the removal recombinant vectors.With restriction enzyme EcoR I and Nsi I digested plasmid carrier pMG425et, with thorough removal erythromycin resistance gene.Carry out enzyme with restriction endonuclease Hinc II again and cut evaluation.In order to identify that further the erythromycin gene in the recombinant plasmid vector determines whether thoroughly cut from the molecular weight angle, the extraction plasmid of positive bacteria is carried out enzyme with restriction endonuclease Hinc II cut evaluation, because according to the physical map of original plasmid vector and the restriction enzyme site of ThyA gene inside: a Hinc II restriction enzyme site is arranged in the inside of erythromycin gene, a Hinc II restriction enzyme site is also arranged in multiple clone site, and also there is a Hinc II restriction enzyme site ThyA gene inside.If so erythromycin gene has been excised fully, after then recombinant vectors is cut by Hinc II enzyme, can only obtain two bar segment, the big fragment of 2657 bases and the small segment of 1059 bases are as Fig. 5.Otherwise, if erythromycin gene is not excised fully, after cutting with Hinc II enzyme, three bar segment have appearred, and wherein a big fragment is 2494 bases, and two small segments are respectively 1059 bases and 1190 bases in addition.
(6) the erythromycin resistance screening is identified.With the positive bacteria large quantity extracting plasmid, cut with restriction enzyme Nsi I enzyme again, reclaim big fragment, reconnect cyclisation, conversion, the real positive bacterium colony of screening, and identify.Identify recombinant vectors containing on the LB substratum of erythromycin: select the male bacterium colony, little extracting plasmid and electrophoresis, screening the less plasmid of molecular weight wherein (being the plasmid of erythromycin gene excision) cultivates containing on the LB substratum of erythromycin, whether cut with definite confirmation erythromycin gene, because if thoroughly excised, then this bacterium just can not grow containing on the LB substratum of erythromycin.Otherwise, then can grow.
(7) evaluation of the repairing effect stability of pMG425t plasmid vector.The intestinal bacteria of ThyA genetic flaw can not grow or poor growth on common LB substratum, must be added with could recover under the thymidine condition of external source the growth.Contain the intestinal bacteria that changing over to of ThyA gene recombination plasmid can make the ThyA genetic flaw and on basic medium, recover growth, the bacterial strain that contains recombinant vectors pMG425t that obtains is continued to go down to posterity not containing on the LB substratum of thymidine, positive colony is extracted plasmid, survey the stability of its reparation.Fig. 6 is for being the lactic acid bacteria shuttle plasmid carrier physics structural representation of selective pressure with ThyA non-antibiotic resistant gene.
Claims (11)
1. non-antibiotic resistance shuttle plasmid expression vector is characterized in that:
At first, original plasmid expression vector pMG26e being carried out enzyme with restriction enzyme cuts; Secondly, at original plasmid expression vector pMG26e polyclone restriction enzyme site situation, design the terminal amplification of specific connection non-antibiotic resistance and select gene NASG; Once more, the non-antibiotic resistance that amplification is obtained selects gene NASG to be connected to specific position; At last, the microbiotic erythromycin gene and the cyclisation of thoroughly removing among the original plasmid vector pMG26e with restriction enzyme obtains plasmid vector pMG425t;
PMG425t does not contain antibiotics resistance gene, and shuttling expressing becomes plasmid expression vector between leather Lan Shi positive bacteria and the cloudy row of leather Lan Shi bacterium.
2. non-antibiotic resistance shuttle plasmid expression vector according to claim 1, it is characterized in that carrying out enzyme at basic plasmid pMG36e restriction enzyme site earlier cuts, ThyA gene in nearly Erythromycin E r end connection non-antibiotic resistant gene NASG is removed the Er gene at last and is obtained charge material grain expression body pMG425t again.
3. non-antibiotic resistance shuttle plasmid expression vector according to claim 1, it is characterized in that carrying out enzyme at basic plasmid pMG36e restriction enzyme site earlier cuts, β-Gal gene in nearly Erythromycin E r end connection non-antibiotic resistant gene NASG is removed the Er gene at last and is obtained plasmid expression vector pMG425t again.
4. a method that makes up the non-antibiotic resistance shuttle plasmid expression vector of claim 1 is characterized in that basic plasmid pMG36e, cuts through restriction enzyme EcoR I enzyme on its restriction enzyme site, becomes line style; Connect non-antibiotic resistant gene NASG, cyclisation winding shape afterwards at nearly Erythromycin E r end again; On this ring, erythromycin gene Er is removed with restriction endonuclease more at last, form non-antibiotic resistance shuttle expression plasmid carrier pMG425t.
5. the method for structure non-antibiotic resistance shuttle plasmid expression vector according to claim 4, it is characterized in that restriction enzyme kind and position according to the antibiotics resistance gene both sides, select to substitute the candidate gene NASG of antibiotics resistance gene, the non-resistance selection markers candidate gene of design primer amplification NASG.
6. the method for structure non-antibiotic resistance shuttle plasmid expression vector according to claim 5 is characterized in that:
1) with restriction endonuclease EcoR I plasmid vector pMG36e is carried out enzyme and cut, make cyclic DNA become line style, handle the plasmid vector that is cut to line style by EcoR I enzyme with polysaccharase T4 DNA again, be connected with the non-antibiotic resistant gene in order to next step;
2) amplification non-antibiotic resistant gene thymidylic acid synthase gene (ThyA) or the beta-galactosidase enzymes (gene of β-Gal), chromosomal DNA with lactobacillus johnsonii or lactobacillus bulgaricus is a template, β-Gal gene order according to lactobacillus johnsonii ThyA gene or lactobacillus bulgaricus, the design primer carries out polymerase chain reaction PCR amplification ThyA or β-Gal gene with high-fidelity polysaccharase Tag PlusI;
3) with 1) in be cut to the basic plasmid pMG36e and 2 of line style) in the non-antibiotic resistant gene ThyA that obtains or (β-gal) be connected, transform, this moment, carrier was for having two resistances, i.e. erythromycin gene and ThyA or (the plasmid vector pMG425et of gene of β-gal);
4) comprise that to having two resistances the plasmid vector pMG425et of erythromycin gene and ThyA gene or β-Gal gene carries out enzyme and cuts with restriction endonuclease EcoR I and Nsi I, thoroughly remove erythromycin resistance gene.
7. the method for structure non-antibiotic resistance shuttle plasmid expression vector according to claim 6, it is characterized in that the ThyA gene or (in the connection procedure of gene of β-gal) and plasmid vector pMG36e, used linear plasmid carrier pMG36e and non-antibiotic resistant gene ThyA or (ratio that is connected of β-gal) is between 1: 1～1: 10.
8. the method for structure non-antibiotic resistance shuttle plasmid expression vector according to claim 6 is characterized in that:
1) the extraction plasmid of positive bacteria is carried out enzyme with restriction endonuclease Hinc II and cut evaluation, determine from the molecular weight angle whether the erythromycin gene the pMG425et is thoroughly cut;
2) recombinant vectors is carried out Function Identification containing on the LB substratum of erythromycin, whether cut with definite confirmation erythromycin gene;
3) determine the ThyA gene among the plasmid vector pMG425t or the repairing effect of β-Gal gene, contain changing over to of ThyA gene or β-Gal gene recombination plasmid and can make the intestinal bacteria of ThyA gene or β-Gal genetic flaw recover growth at basic LB substratum.
9. the purposes of the non-antibiotic resistance shuttle plasmid expression vector of claim 1 is to make milk and milk products, fodder additives and animal vaccine.
10. the purposes of non-antibiotic resistance shuttle plasmid expression vector according to claim 9; it is characterized in that the protective antigen gene of human or animal's disease is connected into plasmid vector; with electric sharp being transformed in the lactobacillus F-strain of carrier, make function milk, sour milk, curdled milk, fermented milk, milk based powders, the milk base leavened prod of anti-specified disease again.
11. the purposes of non-antibiotic resistance shuttle plasmid expression vector according to claim 9; it is characterized in that the protection of animal antigen gene is connected into plasmid vector; again carrier electricity is swashed and be transformed in the lactobacillus F-strain, make specified disease is had the bacterial suspension of prevention, provide protection, dried oral tonic, wet oral tonic, dried tube feed product or wet tube feed product, animality vaccine and has the additive of growth regulating function and trophic function.
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| CN1900289B (en) * | 2006-07-19 | 2010-05-12 | 中国科学院微生物研究所 | Nattokinase expression method and its special expression vector and engineering bacterium |
| CN102010849B (en) * | 2010-11-18 | 2013-09-04 | 广东天浩生物工程股份有限公司 | Recombinant lactic acid bacteria of pig interleukin 10 and application thereof |
| CN102071212A (en) * | 2010-12-09 | 2011-05-25 | 江南大学 | Lactobacillus NICE system and construction method and application thereof |
| CN105974135A (en) * | 2015-11-07 | 2016-09-28 | 张玲 | Kit for detecting nasopharynx cancer and detection method of kit |
| CN113817760B (en) * | 2021-10-13 | 2023-08-22 | 齐鲁工业大学 | Shuttle type food-grade expression vector and construction method and application thereof |
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