CN103484416A - Construction method for engineered strain capable of preparing series of specific salmonella diagnostic serum - Google Patents

Abstract

The invention discloses a construction method for an engineered strain capable of preparing a series of specific salmonella diagnostic serum. The method utilizes a modern molecular biological technology to modify a traditional polyclonal antibody preparation system so as to construct host bacteria lack of surface flagellar antigen synthesis genes; the surface flagellar antigen synthesis genes of different pathogene microbes are cloned on suitable expression vectors, and then the vectors are transferred to the host bacteria lack of the antigen genes, so as to obtain a series of engineered strains which have the same genetic background, and contain specific surface flagellar antigen genes of the different pathogene microbes. Due to the adoption of the technologies, a library containing a plurality of specific salmonella antibodies can be constructed. The antibody library can supplement current antiserum types, and meanwhile can lay foundation for the application of other immunology technologies.

Description

The construction process of one species specificity Salmonellas series diagnosis serum engineering strain

Technical field

The invention belongs to biological product technical field, relate to the engineering strain that can prepare specificity Salmonellas series diagnosis serum, is more specifically a kind of engineering strain and construction process that can prepare specificity Salmonellas H:k diagnostic serum.

Background technology

Salmonellas ( salmonella) belong to enterobacteriaceae ( enterobacteriaceae), be Gram-negative bacteria.Salmonella have specially to the human disease, what have only causes a disease to animal, also has pair humans and animals all to cause a disease.Salmonellosis refer to by salmonella ( salmonella) in different serotypes infect various animals and the general name of the various diseases that causes.Infect the people of Salmonellas or carrier's fecal pollution food, can make the human hair uncooked food poisoning.

Flagellum (flagella) is a kind of accessory structure on gram negative bacterium surface, is responsible for the motion of bacterium.The contained flagellum quantity of the different serotypes of different types of bacterium and same bacterium is also different, from several to tens of, and the longest 70 μ m that reach, diameter is generally 10～20 nm.Except white dysentery Salmonellas and fowl typhoid Salmonellas, Salmonellas has whole body flagellum mostly.By dynamic Salmonellas percutaneous puncture-inoculation, in semisolid medium, it will be along diffusion growth around the puncture alignment.

Salmonellas has complicated antigenic structure, and general Salmonellas has thalline (O) antigen, flagellum (H) antigen and three kinds of antigens of surface antigen (pod membrane or envelope antigen).Flagellar antigen (H antigen) is one of the major antigen on the Gram-negative bacteria surface such as Salmonellas, and the variation on its serology level is significant on somatotype in planting.The flagellin of Salmonellas be by fliCor fljBgenes encoding, about 1.5 kb.The H antigen of Salmonellas is divided into first-phase and second-phase.One by fliCthe coding, two-phase by fljBcoding, these two genes are by phase variation mechanism coordinate expression.Kauffmann-White antigen table is described as much different serotype according to the difference of salmonella O antigen and flagellar antigen by salmonella.At present confirmed that Salmonellas has the serotype more than 2500.

The Salmonellas diagnostic serum is to make inactivated bacteria antigen respectively or the serum of mixed immunity rabbit gained by the representative strain of salmonella, after the nonspecific agglutination element is removed in absorption, makes, and for agglutination test, diagnoses various Salmonellas.Development Salmonellas diagnostic serum is the heat subject of field of biological product always.The method that tradition prepares diagnostic serum comprises the preparation of immunogen and immune serum, and the correlation step such as absorption and calibrating and stability test are very ripe.But traditional method length consuming time, workload is large.And what obtain is polyclonal serum, and its specificity is not high, and some cross reactions often occur.So research and develop new method, be extremely urgent.The present invention prepares the transformation of system to traditional serum, can not only prepare fast the diagnostic serum of high specificity, and can also realize effective removal of cross reaction and stdn and through engineering approaches prepared by serum.

Summary of the invention

The objective of the invention is to build the engineering strain that can prepare specificity Salmonellas H:k diagnostic serum;

Purpose of the present invention is achieved through the following technical solutions:

A kind of engineering strain that can prepare specificity Salmonellas H:k diagnostic serum is characterized in that: it is the recombinant plasmid pTrc99a-that will contain coding flagellar antigen k gene fliC(k), proceed in the Salmonellas of surperficial flagellar antigen synthetic gene disappearance, obtain can effective expression flagellar antigen k engineering strain.

More specifically say by fliCgene is replaced with the chloramphenicol acetyl transferasegene on plasmid pKD3, will fljBgene is replaced with the kalamycin resistance gene on plasmid pKD4, by the microbiotic preliminary screening, goes out deletion mycopremna, then identifies and check order and identify through PCR, obtains corresponding deletion mycopremna G4831 △ fliC△ fljB, and then by the recombinant plasmid pTrc99a-built fliC(k) proceed to wherein, screening obtains engineering strain that can effective expression flagellar antigen k.

The engineering strain that can prepare specificity Salmonellas H:k diagnostic serum of the present invention, a wherein said phase flagellar antigen synthetic gene deletion mycopremna G4831 △ fliC, after disappearance, the gene size is the 1100bp(chloramphenicol acetyl transferasegene), its sequence is SEQ ID NO:1.

The engineering strain that can prepare specificity Salmonellas H:k diagnostic serum of the present invention, wherein said two-phase flagellar antigen synthetic gene deletion mycopremna G4831 △ fliC△ fljB, after disappearance, the gene size is still the 1500bp(kalamycin resistance gene), its sequence is SEQ ID NO:2.

The present invention further discloses the construction process of specificity Salmonellas series diagnosis serum engineering strain, it is characterized in that being undertaken by following step:

(1) surperficial flagellar antigen synthetic gene deletion mycopremna (G4831 △ fliC△ fljB) structure;

(2) recombinant plasmid pTrc99a- fliC(k) structure;

(3) contain recombinant plasmid pTrc99a- fliC(k) structure of clone strain.

The present invention utilizes modern molecular biology technique to prepare system transformed to traditional polyclonal antibody, builds the Host Strains of surperficial flagellar antigen synthetic gene disappearance; The surperficial flagellar antigen synthetic gene of Different Kinds of Pathogens microorganism is cloned in the carrier that is applicable to expressing, and proceed in the Host Strains of antigen gene disappearance, obtain a series of identical genetic background that has, the engineering strain that contains Different Kinds of Pathogens microorganism specific surfaces flagellar antigen gene.With the engineering bacteria antigen-immunized animal of preparation, use and there is identical genetic background but containing the Host Strains absorption of flagellar antigen, do not remove non-purpose antibody, can realize effective removal of cross reaction and stdn and through engineering approaches prepared by antibody.Utilize technology of the present invention to obtain and can prepare specificity H:k, H:r, H:y, H:z, H:z6, H:z10, H:z13, H:z15, H:z28, H:z29, H:v, H:w, the engineering strain of the multiple Salmonellas diagnostic serum such as H:6.

The more detailed construction process of the present invention is as follows: 1, surperficial flagellar antigen synthetic gene deletion mycopremna (G4831 △ fliC△ fljB) structure

The present invention selects this laboratory to be numbered G4831(Ge Luosi to go out the Pu Salmonellas) be the purpose bacterial strain, utilize the Red recombination system mediation flagellin gene of phageλ fliCwith fljBdisappearance.Now for fliCgene illustrates main process, first synthetic pair of primers, 5 of each primer ' end have 39 bp and fliCdNA homolog, 3 ' end and chloramphenicol resistance DNA homolog (for screening), the chloramphenicol acetyl transferasegene of chloramphenicol resistance template plasmid pKD3 (Genebank AY048742) of take is template, in the middle of obtaining by pcr amplification, it is the chloramphenicol acetyl transferasegene of an about 1033bp, the linearity target practice DNA fragmentation that its two ends are the 39bp homology arm, obtain this fragment by cutting glue recovery purifying.This linearity target practice DNA fragment electric shock is transformed in the G4831 that imports in advance the Red recombination system and induces the Red recombination system to express in right amount, lambda exonuclease is attached to linear target practice DNA fragment two 39bp homology arm ends, enzyme is cut and is produced free 3 ' single stranded DNA section, thereby the mode that two homology arms and G4831 interchromosomal are invaded with chain is recombinated.Linear target practice DNA fragment is inserted on G4831 karyomit(e) fliCbetween gene two homology arms, and on G4831 karyomit(e) between two homology arms fliCgene order is replaced by it, thereby has completed fliCthe disappearance of gene, obtain fliCthe genetically deficient bacterial strain.

Red recombination system in the present invention is completed by the pSim plasmid, need to import in advance in the purpose bacterial strain.The pSim plasmid is loaded with the DNA recombination system that is applicable to a lot of intestinal bacteria, is a kind of temperature sensitivity plasmid, in the time of 42 ℃, expresses, and temperature is lost during higher than 32 ℃.

fljBthe disappearance of gene be fliCon genetically deficient bacterial strain basis, another resistance screening fragment of introducing completes, and fliCthe principle of the disappearance of gene is identical.Through above-mentioned twice disappearance, obtain fliCgene and fljBthe bacterial strain that gene all lacks.

2, recombinant plasmid pTrc99a- fliC(k) structure

The present invention is upper relevant with flagellar antigen k by comparison ncbi fliCgene order, through the compare of analysis design fliCgene universal amplification primer, add respectively Nco at 5 of two primers ' end

and BamH restriction enzyme site.

The present invention selects this low copy plasmid of pTrc99a as cloning vector, take this laboratory to be numbered G4816(Thompson Salmonellas) genome is template, uses fliCthe amplification of gene universal amplification primer PCR fliCgene (flagellar antigen k gene), use Nco

and BamH

do double digestion, be connected with the same carrier through double digestion, obtain the recombinant plasmid that contains flagellar antigen k gene.

3, contain recombinant plasmid pTrc99a- fliC(k) structure of clone strain

To cut with PCR and identify that correct recombinant plasmid electricity is transformed into through enzyme fliCgene and fljBin the bacterial strain that gene all lacks, through resistance screening transformant and bacterium colony PCR, identify, obtain the clone strain that contains the recombinant plasmid that can express flagellin k.

4, checking clonal expression experiment

Utilize the expression of bacterium dynamic experiment and Serologic test checking recombinant plasmid.

Bacterium dynamic experiment: the motility principle of specifically utilizing bacterial flagellum, the bacterium of long flagellum can move about in semisolid medium, the bacterium of long flagellum can't not move about, if recombinant plasmid can give expression to flagellin, this flagellin can be assembled into the surface of flagellum disappearance bacterium simultaneously, make it grow flagellum, recover power.The present invention will utilize the expression of the move about situation checking recombinant plasmid of clone strain on semisolid medium.

Serologic test: specifically utilize flagellar antigen to react with the specific binding of corresponding antibodies, bacterium energy amphitrichous and corresponding antibody generation agglutination reaction, do not have bacterium amphitrichous can not, if recombinant plasmid can give expression to flagellin, this flagellin can be assembled into the surface of flagellum disappearance bacterium, makes it grow flagellum.Just can detect with serum the situation that exists of flagellar antigen.The present invention will utilize clone strain and corresponding serum to have or not this situation of agglutination reaction to verify the expression of recombinant plasmid.

The engineering strain that can prepare specificity Salmonellas H:k diagnostic serum prepared by the present invention compared with prior art, has following advantage:

1, feasibility is good: the present invention utilizes the most basic Protocols in Molecular Biology in laboratory to prepare system transformed to traditional polyclonal antibody, by doing disappearance, build the approach such as clone obtain can effective expression flagellin k bacterial strain, thereby obtain specific Salmonellas H:k diagnostic serum, the method practicality, feasible;

2, practical: as by the present invention, to prepare specific Salmonellas H:k diagnostic serum.Compare with traditional method and reduced a large amount of absorption removal work, more save time, and the workload reduction, the serological specificity of production is high, intersects less, greatly reduces the cost of producing serum;

3, handiness is good: utilize technology of the present invention can produce by traditional method and be difficult to the serum of producing, can also build a specific antigen storehouse that comprises several Salmonellas simultaneously.Utilize this antigenic storehouse can set up the antibody library that comprises multiple Salmonellas specific antibody, this antibody library can supplement existing antiserum(antisera) kind, and the application that also can be other immunological techniques simultaneously provides basis.

The accompanying drawing explanation

Fig. 1 is the principle schematic of phageλ Red recombination system used in the present invention;

Fig. 2 is the present invention fliCgenetically deficient bacterium G4831 △ fliCpCR screening electrophoresis result figure; M:DNA Marker (DL2000) in figure; 1, ddH ₂o control; 2, wild-type strain G4831; 3-5, G4831 △ fliCmutants;

Fig. 3 is the present invention fljBgenetically deficient bacterium G4831 △ fliC△ fljBpCR screening electrophoresis result figure; M:DNA Marker (DL2000) in figure; 1, ddH ₂o control; 2, wild-type strain G4831; 3-5, G4831 △ fliC△ fljBmutants;

Fig. 4 is Salmonellas deletion mycopremna G4831 △ prepared by the present invention fliC△ fljB's fliCwith fljBthe genetic stability of disappearance identify, M:DNA Marker (DL2000); 1 and 6, ddH ₂o control; 2 and 7, wild-type strain G4831; 3-5, three generations of G4831 △ fliCmutant; 8-10, three generations of G4831 △ fliC△ fljBmutant;

Fig. 5 is the surperficial flagellar antigen synthetic gene deletion mycopremna G4831 △ that the present invention builds fliC△ fljBgrowth curve;

Fig. 6 is recombinant plasmid pTrc99a-in the present invention fliC(k) schema built;

Fig. 7 is the recombinant plasmid pTrc99a-built in the present invention fliC(k) physical map;

Fig. 8 is the evaluation to recombinant plasmid, wherein: Fig. 8 A: be recombinant plasmid pTrc99a- fliC(k) enzyme is cut qualification result, M:DNA Marker in figure (DL2000 Plus); 1-3, pTrc99a- fliC(k)/Nco

+ BamH

; Fig. 8 B: be recombinant plasmid pTrc99a- fliC(k) PCR qualification result, M:DNA Marker (DL2000) in figure; 1, ddH ₂o control; 2-4, PCR product of pTrc99a- fliC(k);

Fig. 9 is wild-type G4831 of the present invention and G4831 △ fliC△ fljBdisappearance bacterium power plate comparison diagram;

Figure 10 is G4831 △ of the present invention fliC△ fljBdisappearance bacterium and the clone strain G4831 △ that builds fliC△ fljB(pTrc99a- fliC(k)) power plate comparison diagram.

Embodiment

Below in conjunction with Figure of description and specific embodiment, further set forth the present invention.Should understand these embodiment only is not used in and limits the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition as people such as Sambrook, molecular cloning: the condition described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989).The present invention's all ingredients used all has commercially available.

See the following form in bacterial strain uses therefor numbering of the present invention and source:

The laboratory numbering	Former numbering	Chinese	Source
				G4816	50023	The Thompson Salmonellas	CMCC
G4831	50752	Ge Luosi goes out the Pu Salmonellas	CMCC

See the following form in the present invention's plasmid used source:

Embodiment 1:

The structure of deletion mycopremna

First the pSim Plasmid Transformation is advanced to purpose bacterial strain (G4831), then utilize pSim DNA of bacteria recombination system to incite somebody to action fliCgene is replaced with the chloramphenicol acetyl transferasegene on plasmid pKD3, will fljBgene is replaced with the kalamycin resistance gene on plasmid pKD4, by the microbiotic preliminary screening, goes out deletion mycopremna, then identifies and check order and identify through PCR, finally obtains corresponding deletion mycopremna G4831 △ fliC△ fljB.

Concrete step is as follows:

1, fliCthe disappearance of gene

(1) purpose bacterial strain:

Laboratory has bacterial strain G4831 (6,8:z10:e, n, z15) now;

(2) fliCthe design of genetically deficient primer:

Be used for fliCthe chloramphenicol acetyl transferasegene that the primer of gene (about 1.5kb) disappearance be take on pKD3 is pcr template, and adds correspondence at 5 of upstream and downstream primer ' end respectively fliCthe about 39bp of the homologous sequence of gene upstream and downstream, amplification linearity target practice DNA out is about 1.1kb; Described primer (the English Weihe River prompt base (Shanghai) trade Co., Ltd is synthetic) sequence is as follows:

5′-atggcacaagtcattaatacaaacagcctgtcgctgttGTGTAGGCTGGAGCTGCTTCG-3′

5′-ttaacgcagtaaagagaggacgttttgcggaacctggttCATATGAATATCCTCCTTAG-3′

The chosen position of primers designed exists fliCinitial and the terminal position of gene:

5′-ATGGCACAAGTCATTAATACAAAC-3′

5′-TTAACGCAGTAAAGAGAGGACGT-3′

(3) extraction of pKD3 plasmid;

1. from e.colidH5 α – pKD3 glycerine is preserved in pipe and is connect bacterium in 100mL 2YT liquid nutrient medium (Tryptone 16 g, Yeast Extract 10 g, NaCl 5 g, adding distil water is settled to 1 L, 121 ℃ of sterilizing 20 min), in, add penbritin (Sangon Biotech (Shanghai) Co., Ltd.) to final concentration 100 μ g/mL, 37 ℃, 180 r/min, overnight shaking is cultivated; 2. bacterium liquid is joined in 96 deep-well plates to every hole 1ml, 3700rpm, centrifugal 5min; 3. the solution that adds 150 μ L precoolings

(50 mmol/L glucose; 25 mmol/L Tris-HCl; 10 mmol/L EDTA; PH 8.0) and RNaseA(Sangon Biotech (Shanghai) Co., Ltd. of 1 μ L100mg/ml), resuspended thalline; 3. the solution that adds 150 μ L newly to join (1% SDS; 0.2 mol/L NaOH, matching while using), compress slowly upset 10 times, bacterium liquid clear; 4. the solution that adds 150 μ L precoolings (3 mol/L Potassium ethanoates; 2 mol/L acetic acid), white precipitate occurs, compress upset 10 times, be statically placed in 20min in mixture of ice and water, now the precooling whizzer; 5. by deep-well plates in 4 ℃, 3100rcf, centrifugal 30min, get supernatant 300 μ L, turns 2000rpm in 96 hole intercepter plates, 1min is except albumen; 6. add 150 μ L Virahols, fully upset mixes, standing 10min, 3000 rcf, centrifugal 20min, supernatant discarded; 7. add 150 μ L70% washing with alcohol, 3700rpm, centrifugal 5min, supernatant discarded; 8. deep-well plates is inverted on thieving paper to 300 rpm, centrifugal 2s; 9. room temperature is dried, and every hole adds 30 μ L deionized water back dissolvings, is placed in-40 ℃ of refrigerators standby.

(4) take amplification, the product purification that pKD3 is template;

Be used for fliCthe chloramphenicol acetyl transferasegene fragment of genetically deficient be take pKD3 as template amplification, amplification linearity target practice DNA out is about 1.1kb, product, after agarose gel electrophoresis, carries out purifying with cutting glue purification test kit (SanPrep pillar DNA glue reclaims test kit Sangon Biotech (Shanghai) Co., Ltd.) to the fragment of 1.1kb.

(5) conversion, the screening of the preparation of G4831 competent cell and pSim plasmid;

The preparation of G4831 competent cell:

1. preserve pipe and connect bacterium in 10 mL 2YT liquid nutrient mediums from G4831 glycerine, 37 ℃, 180 r/min, overnight shaking is cultivated; 2. the ratio in 1:100 is forwarded in 200 mL 2YT liquid nutrient mediums, and 37 ℃, 250 r/min, shaking culture is to the about 0.4-0.6 of OD600, approximately 2.5 h; 3. bacterium liquid is transferred in 4 50 aseptic mL centrifuge tubes, 4 ℃, 5000 rpm, centrifugal 5min; 4. outwell supernatant, every pipe adds aseptic 10% glycerine of 50 mL (precooling) washing, abundant resuspended precipitation, 4 ℃, 5500 rpm, centrifugal 5 min; 5. outwell supernatant, every pipe adds aseptic 10% glycerine of 25 mL (precooling) washing, and abundant resuspended precipitation, by the synthetic pipe of the bacterium liquid in two pipe centrifuge tubes, 4 ℃, 5500 rpm, centrifugal 5 min; 6. outwell supernatant, every pipe adds aseptic 10% glycerine of 50 mL (precooling) washing, abundant resuspended precipitation, 4 ℃, 5500 rpm, centrifugal 5 min; 7. outwell supernatant, every pipe adds aseptic 10% glycerine of 25 mL (precooling) washing, and abundant resuspended precipitation, by the synthetic pipe of the bacterium liquid in two pipe centrifuge tubes, 4 ℃, 5500 rpm, centrifugal 5 min; 8. outwell supernatant, add aseptic 10% glycerine (precooling) of 800 μ L, abundant resuspended precipitation, divide and be filled in the aseptic centrifuge tube of 0.5 mL, and 80 μ L/pipe, in-80 ℃ of preservations.

The conversion of pSim plasmid, screening:

Get 1 μ L pSim plasmid and join in 80 μ L competent cells, mix gently with the rifle head, the electricity that joins precooling transforms in cup, and then the 1.8KV electric shock joins 200 μ L 2YT at once electricity and transform in cup; Bacterium liquid is coated to the SOB(that contains miewensu (MDBio, Inc) 200 μ g/mL and take Tryptone 20 g, Yeast Extract 5 g, NaCl 0.5 g, KCl 0.186 g, MgCl ₂6H ₂o 2.03 g, adding distil water is settled to 1 L, 105 ℃ of sterilizing 15min, solid SOB substratum adds 1.5% agar powder) on flat board, after 30 ℃ of overnight incubation, choose transformant;

Monoclonal transformant is inoculated in the 4mL SOB liquid nutrient medium that contains miewensu 200 μ g/mL, 30 ℃, 180 r/min, overnight shaking is cultivated;

Extracting plasmid is identified: for identifying correct transformant, get 800 μ L bacterium liquid and add the glycerine that contains the aseptic glycerine of 200 μ L to preserve in pipe, fully mix, be positioned over-80 ℃ of preservations.

(6) conversion of resistance fragment, screening and evaluation;

1. get 10 μ L chloramphenicol acetyl transferasegene amplified productions and join in 80 μ L competent cells, mix gently with the rifle head, the electricity that joins precooling transforms in cup, the 1.8KV electric shock; 2. electricity is transformed to the bacterium liquid sucking-off in cup, join in 1mL SOB substratum, 30 ℃ of 180r/min concussion recovery 2h; 3. bacterium liquid is coated on the SOB flat board that contains paraxin (Sangon Biotech (Shanghai) Co., Ltd.) 15 μ g/mL, after 30 ℃ of overnight incubation, chosen transformant; 4. the picking mono-clonal is inoculated on the SOB flat board that contains paraxin 25 μ g/mL and preserves, and is inoculated in 20 μ L deionized waters, and 99 ℃ are boiled bacterium 15min; 5. the bacterium liquid of getting after heating carries out the PCR evaluation, wild type strain (G4831) in contrast:

Wild type strain (G4831) and fliCdeletion mycopremna (G4831 △ fliC) stripe size is respectively 1.5kb and 1.1kb(specifically is shown in Fig. 2).

(7) the PCR product is carried out to agarose gel electrophoresis, by stripe size, the PCR product of correct bacterial strain carries out purifying with cutting the glue purification test kit, send order-checking to identify;

(8) correct transformant (the G4831 △ that will check order fliC) be inoculated in the 10 mL SOB liquid nutrient mediums that contain paraxin 25 μ g/mL, 37 ℃, 180 r/min, overnight shaking is cultivated;

(9) get 800 μ L bacterium liquid and add the glycerine that contains the aseptic glycerine of 200 μ L to preserve in pipe, fully mix, be positioned over-80 ℃ of preservations.

2, fljBthe disappearance of gene

(1) purpose bacterial strain: above-mentioned structure △ fliCdeletion mycopremna G4831 △ fliC;

(2) for fljBthe kalamycin resistance gene that the primer of gene (about 1.5kb) disappearance be take on pKD4 is pcr template, and adds correspondence at 5 of upstream and downstream primer ' end respectively fljBthe about 39bp of the homologous sequence of gene upstream and downstream; Described primer sequence is as follows:

5′-atggcacaagtaatcaacactaacagtctgtcgctgttGTGTAGGCTGGAGCTGCTTCG-3′

5′-ttaacgtaacagagacagcacgttctgcgggacctggttCATATGAATATCCTCCTTAG-3′

The chosen position of primers designed exists fljBgene zero position and kalamycin resistance gene inside;

5′-ATGGCACAAGTAATCAACAC-3′

5′-CGGTGCCCTGAATGAACTGC-3′

(3) extraction of pKD4 plasmid;

The same

(4) take amplification, the product purification that pKD4 is template;

Be used for fljBgenetically deficient kangene fragment be take pKD4 as template amplification, and amplification linearity target practice DNA out is about 1.5kb, and product, after agarose gel electrophoresis, carries out purifying with cutting the glue purification test kit to the fragment of 1.5kb.

(5) G4831 △ fliCthe conversion of the preparation of competent cell and pSim plasmid, screening;

The same

(6) conversion of resistance fragment, screening and evaluation;

The same

(7) the PCR product is carried out to agarose gel electrophoresis, the correct bacterial strain by stripe size, be PCR with gene two ends primer, with cutting the glue purification test kit, carries out purifying, sending order-checking to identify (specifically seeing sequence table 2);

Wild type strain (G4831) and fljBdeletion mycopremna (G4831 △ fliC△ fljB) stripe size is respectively "-" and 518bp(specifically is shown in Fig. 3).

(8) correct transformant (the G4831 △ that will check order fliC△ fljB) be inoculated in and contain paraxin 25 μ g/mL, in the 10 mL SOB liquid nutrient mediums of kantlex (Sangon Biotech (Shanghai) Co., Ltd.) 50 μ g/mL, 37 ℃, 180 r/min, overnight shaking is cultivated.

(9) get 800 μ L bacterium liquid and add the glycerine that contains the aseptic glycerine of 200 μ L to preserve in pipe, fully mix, be positioned over-80 ℃ of preservations.

Embodiment 2:

Surface flagellar antigen synthetic gene deletion mycopremna G4831 △ fliC△ fljBbiological property analysis

(1) surperficial flagellar antigen synthetic gene deletion mycopremna G4831 △ fliC△ fljBthe genetic stability analysis

Deletion mycopremna is passed continuously on the LB solid medium to 10 generations, every Dai Douyong fliC, fljBprimers designed is identified genetic stability, and result shows continuous 10 generations that passed, and all can only amplify the fragment of 1.1kb and 518bp.Salmonellas deletion mycopremna G4831 △ prepared by the present invention is described fliC△ fljB's fliCwith fljBvery stable (specifically the seeing Fig. 4) of disappearance.

(2) surperficial flagellar antigen synthetic gene deletion mycopremna G4831 △ fliC△ fljBthe growth characteristics analysis

Deletion mycopremna G4831 △ fliC△ fljBpresent obvious motionless state (specifically seeing Fig. 9) on 0.3% semisolid flat board.And can not swim over to the other end from an end of U-shaped pipe.Wild type strain and deletion mycopremna are connected to respectively in the LB liquid nutrient medium and cultivate 24 hours, every sampling in a hour, survey OD600 value, the drafting growth curve.The result demonstration, deletion mycopremna and wild type strain growing state basically identical (specifically seeing Fig. 5), illustrate that the disappearance of flagellin gene is little to the growth effect of bacterium, the growth of deletion mycopremna is very stable.

Embodiment 3:

Recombinant plasmid pTrc99a- fliC(k) structure

1, flagellin antigen k, fliCgene, amplification is existing bacterial strain G4816 (6,7:k:1,5) from laboratory.

2, due to fliCgene two ends high conservative, download relevant flagellar antigen k gene from ncbi, through comparison, and design fliCgene universal amplification primer.

3, cloning vector: plasmid pTrc99a, the restriction enzyme site of selection: Nco

and BamH

.

4, PCR amplifying target genes.

1) amplimer: Nco is designed respectively at two ends

and BamH

restriction enzyme site, amplification length 1.5kb left and right;

Primer1：5′-CATG CCATGGCACAAGTCATTAATACAAAC-3′ (Nco

)

Primer2：5′-CG GGATCCTTAACGCAGTAAAGAGAGGACGT-3′ (BamH )

2) PCR system (50 μ L):

10× Pfu buffer with MgSO ₄ (Thermo Scientific) 5μL

MgSO ₄ (Thermo Scientific) 2μL

dNTP 4μL

genome (G4816) 1μL

Primer1 1μL

Primer2 1μL

Pfu DNA Polymerase (Thermo Scientific) 0.5μL

ddH ₂O 35.5μL

3) PCR program:

95℃ 3min

95℃ 30s

45℃ 30s

72 ℃ of 3min30s are totally 30 cycles

72℃ 10min

4℃

5, the PCR product is cut the glue recovery

6, enzyme is cut

Reaction system (30 μ L)

10×NEB Buffer 3.1 (NEB) 3μL

fliC(k) 25.2μL

Nco

(NEB) 0.75μL

BamH

(NEB) 0.75μL

10×BSA (NEB) 0.3μL

10×NEB Buffer 3.1 (NEB) 3μL

pTrc99a 25.2μL

Nco

(NEB) 0.75μL

BamH

(NEB) 0.75μL

10×BSA (NEB) 0.3μL

37 ℃ of enzymes are cut and are spent the night

7, to cut the product post pure for the goal gene enzyme, and the plasmid enzyme restriction product is cut glue and reclaimed, electrophoresis detection concentration:

SanPrep pillar PCR product purification test kit (Sangon Biotech (Shanghai) Co., Ltd.)

8, connect

Linked system (20 μ L)

T4 DNA Ligase (Promega) 1μL

Ligase 10×Buffer (Promega) 2μL

pTrc99a 3μL

fliC(k) 14μL

Room temperature connects 3-4 hour

9,8 uL connection products add electricity in 80 uLDH5 α competence to turn, add and in the every 100 mL SOB substratum of 1mL SOC(, add the 1M glucose solution to mix to be) recovery 1 hour in substratum, be coated with Amp resistant panel, 37 ℃ of overnight incubation after the centrifugal 5min of 4000rpm.

10, the picking mono-clonal connects 96 orifice plates (2YT+Amp), 180r/min, 37 ℃ of overnight incubation.

11, extract plasmid pTrc99a- fliC(k).

12, enzyme is cut evaluation.

The enzyme slitting band size of recombinant plasmid is respectively 4.1kb and 1.5kb left and right (specifically seeing Fig. 8).

Embodiment 4

The structure of clone strain

1, preparation G4831 △ fliC△ fljBcompetent cell.

2,1 uL pTrc99a- fliC(k) add 80 uL G4831 △ fliC△ fljBin competence, electricity turns, and adds in 1mL SOC substratum and recovers 1 hour, is coated with Amp resistant panel, 37 ℃ of overnight incubation after the centrifugal 5min of 4000rpm.

3, picking mono-clonal bacterium colony PCR identifies.

4, protect bacterium G4831 △ fliC△ fljB(pTrc99a- fliC(k)).

Embodiment 5:

Serologic test

1, checking clonal expression experiment

(1) get the bacterium G4831 △ in the glycerine pipe fliC△ fljB(pTrc99a- fliC(k)) 3 uL are connected to respectively in 2 parallel U-shaped pipes that fill 0.5% semisolid medium, put 37 ℃ of incubated overnight, and this bacterium all can grow to the other end from an end of U-shaped pipe, G4831 △ fliC△ fljBcan not;

(2) get the bacterium G4831 △ in the glycerine pipe fliC△ fljB(pTrc99a- fliC(k)) 1 uL is connected to respectively in the semisolid flat board of 2 parallel 0.3%, 37 ℃ of incubated overnight, and the equal amphitrichous of the bacterium colony on flat board grows (specifically seeing Figure 10), G4831 △ fliC△ fljBdo not have.

2, serum confirmatory experiment

Draw the bacterium liquid 5 μ L that grow to the U-shaped pipe the other end in (1), or in picking (2), the flagellum on semi-solid flat board is transferred when 5mL shakes the mid-37 ℃ of static OD of the being cultured to values of pipe for 0.4-0.6,5500rpm, centrifugal 5min receives bacterium, after outwelling supernatant, add the vibration of 200uL stroke-physiological saline solution resuspended.

Slide agglutination test: draw serum 10 μ L on aseptic slide glass, add the resuspended bacterium liquid of 5 μ L in serum, mix, the aggegation result that can detect by an unaided eye in a minute, be 3+ and above agglutination phenomenon is positive, and attention will be with PBS solution in contrast.

Adopt respectively clone strain that the present invention builds and traditional method slide agglutination test contrast:

Conclusion:

(1) clone strain with salmonellaagglutination reaction occurs in H:k, and the reaction clear, and agglutinating particle is more, larger, and response intensity is 4+, and with salmonellah:1 complex and salmonellaany agglutination reaction does not occur in H:5;

(2), according to " the salmonella diagnostic serum is manufactured and vertification regulation " in " Products in China rules " (version in 2000), the preparation process of serum is divided into the preparation of immunizing antigen, immunizing antigen calibrating, immunity, blood sampling, with immune bacterium, measures serum titer, absorbs the steps such as the non-purpose antibody of removal and finally obtain serum.The present invention uses respectively engineering bacteria G4831 △ fliC△ fljB(pTrc99a- fliC) and traditional method G4816 (6 (k), 7:k:1,5) after above step immunizing rabbit, take a blood sample, separation of serum, in absorbing this step, the serum be separated to engineering bacterial immunity rabbit absorbs bacterium with corresponding diphasic antigen agglutination reaction occurs hardly, and the serum obtained by traditional method need just can obtain specific antiserum(antisera) k through a large amount of absorption work;

(3) utilize technology of the present invention can obtain specific Salmonellas diagnostic serum, and consuming time short, cost is low.

Embodiment 5: application example

Utilize technology of the present invention can make most Salmonellas H diagnostic antigen serum:

These serum can be used for the evaluation of Salmonellas H antigen, also can be used for the gram negative bacillus separated by patient's ight soil or other materials, and through preliminary biochemical analysis, doubtful is the evaluation of the bacterial strain of salmonella.

Concrete using method is as follows: bacterium through the semisolid medium puncture once, picking has grown into the bacterium colony at substratum top, be transferred on solid medium, incubated overnight, first do slide agglutination test with various polyvalent serums respectively, during positive reaction, then do slide agglutination test with corresponding monovalent serum, make diagnosis.Drip two physiological saline separated from one another with transfering loop on clean slide.

From nutrient agar plate, picking divides pure bacterium colony, respectively with slide on physiological saline mix.

With transfering loop drip 1～2 bleed clearly (10-15uL) in the mixed solution of bacterium colony and physiological saline, add a physiological saline in the mixed solution of another bacterium colony and physiological saline in contrast on slide.

Mix respectively two kinds of mixed solutions on slide with transfering loop.

Shake gently slide 1 minute, observe.Be 2+ and above agglutination phenomenon is positive in 1 minute, present the following agglutination phenomenon of 2+ in 1 minute negative.

Aggegation judging criterion as a result is as follows:

Response intensity	Distinguishing rule
			4+	The reaction clear, agglutinating particle is more, larger
3+	The reaction clear, agglutinating particle is more, less
		2+	React some muddiness, but still visible more particulate state aggegation
1+	React very muddy, only have a little particulate state aggegation
		-	Any aggegation, do not appear in the muddiness that reacts completely

SEQUENCE LISTING

<110 > Nankai University

<120 > construction process of a species specificity Salmonellas series diagnosis serum engineering strain

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1112

<212> DNA

<213 > artificial sequence

<400> 1

catgccatgg cacaagtcat taatacaaac agcctgtcgc tgttgtgtag gctggagctg 60

cttcgaagtt cctatacttt ctagagaata ggaacttcgg aataggaact tcatttaaat 120

ggcgcgcctt acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt 180

ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag cggcatcagc 240

accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc 300

atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa 360

aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca 420

tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat 480

gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc 540

accagctcac cgtctttcat tgccatacgt aattccggat gagcattcat caggcgggca 600

agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag 660

gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc 720

tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt 780

ttctccattt tagcttcctt agctcctgaa aatctcgaca actcaaaaaa tacgcccggt 840

agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc aacgtctcat 900

tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg atttatttat 960

tctgcgaagt gatcttccgt cacaggtagg cgcgccgaag ttcctatact ttctagagaa 1020

taggaacttc ggaataggaa ctaaggagga tattcatatg aaccaggttc cgcaaaaacg 1080

tcctctcttt actggcgtta aaggatcccg ga 1112

<210> 2

<211> 1537

<212> DNA

<213 > artificial sequence

<400> 2

tatgccatgg cacaagtaat caacactaac agtctgtcgc tgttgtgtag gctggagctg 60

cttcgaagtt cctatacttt ctagagaata ggaacttcgg aataggaact tcaagatccc 120

ccacgctgcc gcaagcactc agggcgcaag ggctgctaaa ggaagcggaa cacgtagaaa 180

gccagtccgc agaaacggtg ctgaccccgg atgaatgtca gctactgggc tatctggaca 240

agggaaaacg caagcgcaaa gagaaagcag gtagcttgca gtgggcttac atggcgatag 300

ctagactggg cggttttatg gacagcaagc gaaccggaat tgccagctgg ggcgccctct 360

ggtaaggttg ggaagccctg caaagtaaac tggatggctt tcttgccgcc aaggatctga 420

tggcgcaggg gatcaagatc tgatcaagag acaggatgag gatcgtttcg catgattgaa 480

caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac 540

tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg 600

cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag 660

gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt 720

gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg 780

tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg 840

catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga 900

gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag 960

gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat 1020

ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt 1080

tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg 1140

gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt 1200

tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc 1260

ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac 1320

gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg 1380

acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccaccccc 1440

cagctttcaa aaaagcgctc ctgaagttcc tatacttttc tagagaatag gaacttcggg 1500

aataggaact aaggaggatt ttcatatgaa ccaggtc 1537

Claims (5)

1. the engineering strain that can prepare specificity Salmonellas H:k diagnostic serum is characterized in that: it is the recombinant plasmid pTrc99a-that will contain coding flagellar antigen k gene fliC(k), proceed to the Salmonellas G4831 △ of surperficial flagellar antigen synthetic gene disappearance fliC△ fljBin obtain can effective expression flagellar antigen k engineering strain.

2. the engineering strain that can prepare specificity Salmonellas H:k diagnostic serum claimed in claim 1 is characterized in that: it be by fliCgene is replaced with the chloramphenicol acetyl transferasegene on plasmid pKD3, will fljBgene is replaced with the kalamycin resistance gene on plasmid pKD4, by the microbiotic preliminary screening, goes out deletion mycopremna, then identifies and check order and identify through PCR, obtains corresponding deletion mycopremna G4831 △ fliC△ fljB, and then by the recombinant plasmid pTrc99a-built fliC(k) proceed to wherein, screening obtains engineering strain that can effective expression flagellar antigen k.

3. the engineering strain that can prepare specificity Salmonellas H:k diagnostic serum claimed in claim 2, a wherein said phase flagellar antigen synthetic gene deletion mycopremna G4831 △ fliC, after disappearance, the gene size is about the 1100bp(chloramphenicol acetyl transferasegene), its sequence is SEQ ID NO:1.

4. the engineering strain that can prepare specificity Salmonellas H:k diagnostic serum claimed in claim 2, wherein said two-phase flagellar antigen synthetic gene deletion mycopremna G4831 △ fliC△ fljB, after disappearance, the gene size is about the 1500bp(kalamycin resistance gene), its sequence is SEQ ID NO:2.

5. the construction process of a species specificity Salmonellas series diagnosis serum engineering strain is characterized in that carrying out as follows:

(1) surperficial flagellar antigen synthetic gene deletion mycopremna (G4831 △ fliC△ fljB) structure;

(2) recombinant plasmid pTrc99a- fliC(k) structure;

(3) contain recombinant plasmid pTrc99a- fliC(k) structure of clone strain.

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