CN103623399A - Method for preparing and identifying recombinant bifidobacterium vaccine of taenia solium TSO45W-4B gene - Google Patents
Method for preparing and identifying recombinant bifidobacterium vaccine of taenia solium TSO45W-4B gene Download PDFInfo
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Abstract
The invention discloses a method for preparing and identifying a recombinant bifidobacterium vaccine of a taenia solium TSO45W-4B gene. The taenia solium TSO45W-4B gene is synthetized by a complete genome, and directionally cloned to an escherichia coli-bifidobacterium expression vector pGEX-1lambdaT; a recombinant plasmid pGEX-TSO45W-4B is built; bifidobacterium longum (B.longum) is transformed in a manner of electroporation, and a taenia solium recombinant Bb(pGEX-TSO45W-4B) vaccine is built. Thus, a valuable novel vaccine is provided for prevention and treatment of cysticercosis cellulosae.
Description
Technical field
The present invention relates to the preparation of taeniasis suis TSO45W-4B gene recombinaton bacillus bifidus vaccine and authentication method.
Background technology
Cysticercosis cellulosae (Cysticercosis cellulosae) claim again cysticercosis (Cysticercosis), it is the infecting both domestic animals and human parasitic disease of a kind of serious harm human health that the internal organs such as silk ribbon phase parasitized larvae, muscle subcutaneous at human body, brain, eye cause in taeniasis suis (Taenia solium), become global public health problem, China is also one of cysticercosis cellulosae country occurred frequently, higher with northeast, North China, northwest, southwest sickness rate, approximately there are 2,000,000～3,000,000 Patients With Cysticercosis in the whole nation, and infection rate is 0.14%～3.20%.Medicine and operative treatment have its limitation, and this disease of control that develops vaccine has become current research focus.
TSO45W-4B gene is important immunogen gene of Taenia solium oncosphere stage, is considered to the Vaccine candidate gene of tool prospect.Bacillus bifidus (Bifidobacteria, Bb) is important probiotic bacteria in people and mammal intestinal, has the multiple physiological actions such as antibacterial, tumor suppression, immunomodulating and nutrition.In recent years, along with the development of technique for gene engineering, take Bb as carrier transfer system, in fields such as antibacterial, virus, tumors, built a series of recombined bifidobacterias.And aspect parasite field, there is not yet the report of taeniasis suis restructuring Bb, therefore, this research is intended by the synthetic taeniasis suis TSO45W-4B gene of full gene, by its directed cloning in Escherichia coli-Bifidobacterium shuttle expression carrier pGEX-1 λ T, construction recombination plasmid pGEX-TSO45W-4B, electroporation transforms bifidobacterium longum (B.longum), build taeniasis suis restructuring Bb (pGEX-TSO45W-4B) vaccine, for the control of cysticercosis cellulosae provides a kind of valuable new generation vaccine.
Summary of the invention
The technical problem to be solved in the present invention is: by the synthetic taeniasis suis TSO45W-4B gene of full gene, by its directed cloning in Escherichia coli-Bifidobacterium shuttle expression carrier pGEX-1 λ T, construction recombination plasmid pGEX-TSO45W-4B, electroporation transforms bifidobacterium longum (B.longum), build taeniasis suis restructuring Bb (pGEX-TSO45W-4B) vaccine, for the control of cysticercosis cellulosae provides a kind of valuable new generation vaccine.
Technical scheme of the present invention is: a kind of taeniasis suis TSO45W-4B gene recombinaton bacillus bifidus vaccine preparation and authentication method, comprise the following steps:
(1) TSO45W-4B gene synthesizes and design of primers: according to taeniasis suis TSO45W-4B gene order, by the synthetic method of full gene, design total length splicing primer, at the two ends of primer, add respectively BamH I and EcoR I restriction enzyme site and protectiveness base, the TSO45W-4B gene that composition length is 351bp;
(2) structure of recombiant plasmid pGEX-TSO45W-4B and evaluation: the PCR product of synthetic gene and cloning vehicle pMD18-T are under the effect of T4DNA ligase, connect, connect product and be converted into E.coli Top10 competent cell, ice bath 30min, 42 ℃ of water-bath 90s, after ice bath is cooling, add LB culture fluid, jolting, centrifugal, abandon supernatant, remain 200 μ l and mix on the LB agar plate being applied to containing Amp, be inverted and cultivate 12～16h; Picking list bacterium colony is to containing in the LB culture fluid of ampicillin, and jolting is cultivated, and extracts plasmid, carries out PCR evaluation; Then recombiant plasmid pMD18-T-TSO45W-4B and expression vector pGEX-1 λ T are used respectively to BamH I and EcoR I double digestion, glue connects with T4DNA ligase after reclaiming respective segments, be converted into E.coli DH5 α competent cell, picking positive colony, extracting plasmid, carries out enzyme action and order-checking is identified;
(3) structure and the evaluation of taeniasis suis restructuring Bb (pGEX-TSO45W-4B) vaccine
1. the preparation of B.longum competence antibacterial: after B.longum lyophilized powder is fully dissolved with sterilized water, coat on MRS agar plate, anaerobism is cultivated 24～72h, and it is fully activated; Single bacterium colony of picking activation, is inoculated in anaerobism in MRS liquid culture and cultivates; Then in 1:25 ratio, be inoculated in the MRS culture medium containing sucrose, anaerobism is cultivated 24～72h; Ice bath, centrifugal, abandon supernatant, precipitation is cleaned with the sucrose of pre-cooling, then uses sucrose buffer resuspended; This antibacterial can transform for electricity immediately, or adds the glycerol of pre-cooling, and-80 ℃ frozen standby;
2. recombiant plasmid pGEX-TSO45W-4B electricity transforms B.longum competence antibacterial: get standby recombiant plasmid pGEX-TSO45W-4B and B.longum competence antibacterial mixes, after ice bath 10～15min, be transferred in electric shock cup, after electric shock, immediately mixed liquor is transferred in the EP pipe of MRS fluid medium, anaerobism is cultivated; Bacterium liquid is coated on the MRS agar plate containing ampicillin, and anaerobism is cultivated 24～72h;
3. the evaluation of taeniasis suis restructuring Bb (pGEX-TSO45W-4B) vaccine: in the above-mentioned flat board of picking, single bacterium colony is to the EP pipe of the MRS culture medium containing ampicillin, and anaerobism is cultivated 24～72h; Bacterium liquid is centrifugal, abandon supernatant, in precipitation, add the heavy lysosome precipitation of sucrose solution, temperature is bathed, during vibration it is fully reacted; Extracting plasmid, carries out enzyme action, PCR and order-checking and identifies.
the beneficial effect that the present invention reaches: by the synthetic taeniasis suis TSO45W-4B gene of full gene, by this gene directed cloning in Escherichia coli-Bifidobacterium shuttle expression carrier pGEX-1 λ T, construction recombination plasmid pGEX-TSO45W-4B, electroporation imports Bb by this plasmid, build taeniasis suis restructuring Bb (pGEX-TSO45W-4B) vaccine, by enzyme action, PCR and order-checking, identify, prove and successfully built taeniasis suis restructuring Bb (pGEX-TSO45W-4B) vaccine.Vaccine of the present invention is a kind of new generation vaccine, has many advantages: 1. the restructuring Bb vaccine of structure is live vaccine, long-term surviving constantly expression exogenous antigen in vivo, and single inoculation gets final product the reaction of successive induction to target antigen, produces lasting immunne response; 2. use molecular biology method, by the shearing of target antigen or modification, can make new generation vaccine idealized; 3. Bb itself has physiologically active widely, and therefore, as in vaccine application, the prebiotic function that Bb can performance itself has, reaches the multiplex object of potion; 4. produce simply, expressed protective antigen does not need purification, without adjuvant, can be directly used in immunity inoculation, has removed the complicated procedures of forming of protein post processing from, thereby greatly reduces the cost of vaccine, is suitable for producing in enormous quantities; 5. route of inoculation is convenient, can adopt collunarium, the acceptant approach of crowd such as oral to carry out immunity; 6. in view of Bb has generally acknowledged safety and unique good to eat local flavor, vaccine can be applied by the method for adding in the beverages such as Yoghourt, is expected a kind of safe and reliable brand-new means and the approach that become numerous crowds' prevention, control cysticercosis cellulosae.Therefore, selecting the bacillus bifidus (Bb) of Body normal flora is carrier, build the restructuring Bb vaccine of taeniasis suis, may be prevention and safer, the effective a kind of new generation vaccine of control cysticercosis cellulosae, the control that this vaccine is used for to people from Endemic Area cysticercosis cellulosae has great Social benefit and economic benefit.
Accompanying drawing explanation
Fig. 1 is that the double digestion of recombiant plasmid pGEX-TSO45W-4B is identified, wherein 1: the double digestion product of recombiant plasmid pGEX-TSO45W-4B; 2:DNA molecular mass standard;
Fig. 2 is that the order-checking of recombiant plasmid pGEX-TSO45W-4B is identified;
Fig. 3 is that the double digestion of recombiant plasmid pGEX-TSO45W-4B in B.longum is identified, wherein M:DNA molecular mass standard; 1: recombiant plasmid pGEX-TSO45W-4B; 2: the double digestion product of recombiant plasmid pGEX-TSO45W-4B;
Fig. 4 is that the PCR of recombiant plasmid pGEX-TSO45W-4B in B.longum identifies, wherein M:DNA molecular mass standard; 1～8: the PCR product of recombiant plasmid pGEX-TSO45W-4B in bacillus bifidus;
Fig. 5 is that the order-checking of recombiant plasmid pGEX-TSO45W-4B in B.longum is identified.
The specific embodiment
Taeniasis suis TSO45W-4B gene recombinaton bacillus bifidus vaccine preparation of the present invention and authentication method, comprise the following steps:
(1) TSO45W-4B gene synthesizes and design of primers: according to taeniasis suis TSO45W-4B gene order, by the synthetic method of full gene, design total length splicing primer, adds respectively BamH I and EcoR I restriction enzyme site and protectiveness base at the two ends of primer.The TSO45W-4B gene that is 351bp by the handsome Bioisystech Co., Ltd in Shanghai composition length.
(2) structure of recombiant plasmid pGEX-TSO45W-4B and evaluation: the PCR product of synthetic gene and cloning vehicle pMD18-T are under the effect of T4DNA ligase, 16 ℃ of connections are spent the night, connect product and be converted into E.coli Top10 competent cell, ice bath 30min, 42 ℃ of water-bath 90s, after ice bath is cooling, add 900 μ lLB culture fluid, 37 ℃ of 225rpm jolting 45 min, centrifugal, abandon supernatant 400 μ l left and right, remain 200 μ l and mix and be applied to containing on the LB agar plate of 50 μ g/mlAmp, be inverted for 37 ℃ and cultivate 12～16h.Picking list bacterium colony is to containing in the LB culture fluid of 50 μ g/ml ampicillin, and 37 ℃ of 225rpm jolting overnight incubation, extract plasmid, carry out PCR evaluation.Then recombiant plasmid pMD18-T-TSO45W-4B and expression vector pGEX-1 λ T are used respectively to BamH I and EcoR I double digestion, glue spends the night with 16 ℃ of connections of T4DNA ligase after reclaiming respective segments, be converted into E.coli DH5 α competent cell, picking positive colony, extracting plasmid, carries out enzyme action and order-checking is identified.
The enzyme action of recombiant plasmid pGEX-TSO45W-4B is identified:
Recombiant plasmid pGEX-TSO45W-4B warp
bamH Iwith
ecoR Idouble digestion, uses 1% agarose gel electrophoresis, obtains the pGEX-1 λ T carrier segments of 4944 bp and the TSO45W-4B genetic fragment of 351bp, conforms to expected results, sees Fig. 1.
The order-checking of recombiant plasmid pGEX-TSO45W-4B is identified
Synthetic TSO45W-4B gene, is subcloned into BamH I and the EcoR I site of pGEX-1 λ T carrier, and sequencing result mates with expected sequence 100%, sees Fig. 2.
(3) structure and the evaluation of taeniasis suis restructuring Bb (pGEX-TSO45W-4B) vaccine
1. the preparation of B.longum competence antibacterial: after the B.longum lyophilized powder of purchase is fully dissolved with sterilized water, coat on MRS agar plate, 37 ℃ of anaerobism are cultivated 24～72h, and it is fully activated; Single bacterium colony of picking activation, is inoculated in 4mlMRS liquid culture, and it is 0.5 left and right that 37 ℃ of anaerobism are cultured to thalline OD600nm value; In 1:25 ratio, be inoculated in the MRS culture medium containing 0.5M sucrose, 37 ℃ of anaerobism are cultivated 24～72h; Ice bath 30min, 4 ℃ of centrifugal 1min of 10000r/min, abandon supernatant, and precipitation is cleaned 2 times with the 0.5M sucrose of pre-cooling, then uses 100 μ l0.5M sucrose buffer resuspended; This antibacterial can transform for electricity immediately, or adds 15% glycerol of pre-cooling, and-80 ℃ frozen standby.
2. recombiant plasmid pGEX-TSO45W-4B electricity transforms B.longum competence antibacterial: get standby recombiant plasmid pGEX-TSO45W-4B1 μ g and B.longum competence antibacterial 100 μ l mix, after ice bath 10～15min, be transferred to the electric shock cup of 1mm, shock parameters is set to: voltage 1.25KV, field intensity 12.5 KV/cm, electric capacity 25 μ F, resistance 200 Ω, transformation time 5ms; After electric shock, immediately mixed liquor is transferred in the EP pipe of 900 μ lMRS fluid mediums, 37 ℃ of anaerobism are cultivated 2h; Bacterium liquid is coated containing on the MRS agar plate of 100 μ g/ml ampicillin, and 37 ℃ of anaerobism are cultivated 24～72h.
3. the evaluation of taeniasis suis restructuring Bb (pGEX-TSO45W-4B) vaccine: in the above-mentioned flat board of picking, single bacterium colony is to the EP pipe of 1ml containing the MRS culture medium of 100 μ g/ml ampicillin, and 37 ℃ of anaerobism are cultivated 24～72h; By 4 ℃ of centrifugal 5min of 10000r/min of bacterium liquid, abandon supernatant, in precipitation, add the heavy lysosome precipitation of 250 μ l 25% sucrose solutions, 37 ℃ of temperature are bathed 30min, during every 5min vibration 1 time, it is fully reacted; Extracting plasmid, carries out enzyme action, PCR and order-checking and identifies.
In B.longum, the enzyme action of recombiant plasmid pGEX-TSO45W-4B is identified:
The recombiant plasmid pGEX-TSO45W-4B of extracting from there is the B.longum of amicillin resistance, through BamH I and EcoR I double digestion, use 1% agarose gel electrophoresis, obtain the pGEX-1 λ T carrier segments of 4944 bp and the TSO45W-4B genetic fragment of 351bp, conform to expected results, see Fig. 3.
b.longumthe PCR of middle recombiant plasmid pGEX-TSO45W-4B identifies
With from thering is amicillin resistance
b.longumthe recombiant plasmid pGEX-TSO45W-4B of middle extracting is that template is carried out the genetic fragment that pcr amplification can obtain 450bp, removes carrier sequence 99bp, and the length of TSO45W-4B gene reality is 351bp, conforms to expected results, sees Fig. 4.
In B.longum, the order-checking of recombiant plasmid pGEX-TSO45W-4B is identified:
The recombiant plasmid pGEX-TSO45W-4B of extracting from have the B.longum of amicillin resistance, its sequencing result mates with expected sequence 100%, sees Fig. 5.
Claims (2)
1. taeniasis suis TSO45W-4B gene recombinaton bacillus bifidus vaccine is prepared and an authentication method, it is characterized in that: comprise the following steps:
(1) TSO45W-4B gene synthesizes and design of primers: according to taeniasis suis TSO45W-4B gene order, by the synthetic method of full gene, design total length splicing primer, at the two ends of primer, add respectively BamH I and EcoR I restriction enzyme site and protectiveness base, the TSO45W-4B gene that composition length is 351bp;
(2) structure of recombiant plasmid pGEX-TSO45W-4B and evaluation: the PCR product of synthetic gene and cloning vehicle pMD18-T are under the effect of T4DNA ligase, connect, connect product and be converted into E.coli Top10 competent cell, ice bath 30min, 42 ℃ of water-bath 90s, after ice bath is cooling, add LB culture fluid, jolting, centrifugal, abandon supernatant, remain 200 μ l and mix on the LB agar plate being applied to containing Amp, be inverted and cultivate 12～16h; Picking list bacterium colony is to containing in the LB culture fluid of ampicillin, and jolting is cultivated, and extracts plasmid, carries out PCR evaluation; Then recombiant plasmid pMD18-T-TSO45W-4B and expression vector pGEX-1 λ T are used respectively to BamH I and EcoR I double digestion, glue connects with T4DNA ligase after reclaiming respective segments, be converted into E.coli DH5 α competent cell, picking positive colony, extracting plasmid, carries out enzyme action and order-checking is identified;
(3) structure and the evaluation of taeniasis suis restructuring Bb (pGEX-TSO45W-4B) vaccine
1. the preparation of B.longum competence antibacterial: after B.longum lyophilized powder is fully dissolved with sterilized water, coat on MRS agar plate, anaerobism is cultivated 24～72h, and it is fully activated; Single bacterium colony of picking activation, is inoculated in anaerobism in MRS liquid culture and cultivates; Then in 1:25 ratio, be inoculated in the MRS culture medium containing sucrose, anaerobism is cultivated 24～72h; Ice bath, centrifugal, abandon supernatant, precipitation is cleaned with the sucrose of pre-cooling, then uses sucrose buffer resuspended; This antibacterial can transform for electricity immediately, or adds the glycerol of pre-cooling, and-80 ℃ frozen standby;
2. recombiant plasmid pGEX-TSO45W-4B electricity transforms B.longum competence antibacterial: get standby recombiant plasmid pGEX-TSO45W-4B and B.longum competence antibacterial mixes, after ice bath 10～15min, be transferred in electric shock cup, after electric shock, immediately mixed liquor is transferred in the EP pipe of MRS fluid medium, anaerobism is cultivated; Bacterium liquid is coated on the MRS agar plate containing ampicillin, and anaerobism is cultivated 24～72h;
3. the evaluation of taeniasis suis restructuring Bb (pGEX-TSO45W-4B) vaccine: in the above-mentioned flat board of picking, single bacterium colony is to the EP pipe of the MRS culture medium containing ampicillin, and anaerobism is cultivated 24～72h; Bacterium liquid is centrifugal, abandon supernatant, in precipitation, add the heavy lysosome precipitation of sucrose solution, temperature is bathed, during vibration it is fully reacted; Extracting plasmid, carries out enzyme action, PCR and order-checking and identifies.
2. taeniasis suis TSO45W-4B gene recombinaton bacillus bifidus vaccine according to claim 1 preparation and authentication method, is characterized in that: step 2. described in shock parameters be set to: voltage 1.25KV, field intensity 12.5 KV/cm, electric capacity 25 μ F, resistance 200 Ω, transformation time 5ms.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104099364A (en) * | 2014-07-15 | 2014-10-15 | 遵义医学院 | Method for researching stability of recombinant bifidobacteria vaccine for taenia solium |
| CN112891523A (en) * | 2021-03-25 | 2021-06-04 | 遵义医科大学 | Preparation and identification method of taenia solium Ts14-3-3.3 DNA vaccine |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0552850A1 (en) * | 1984-10-10 | 1993-07-28 | Centocor, Inc. | Cloning and expression of HTLV-III DNA |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0552850A1 (en) * | 1984-10-10 | 1993-07-28 | Centocor, Inc. | Cloning and expression of HTLV-III DNA |
Non-Patent Citations (2)
| Title |
|---|
| 周必英等: "猪带绦虫TSO45W-4B基因的克隆、表达和抗体制备", 《中国寄生虫学与寄生虫病杂志》 * |
| 周必英等: "细粒棘球绦虫重组Bb-Eg95-EgA31融合基因疫苗构建及鉴定", 《中国人兽共患病学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104099364A (en) * | 2014-07-15 | 2014-10-15 | 遵义医学院 | Method for researching stability of recombinant bifidobacteria vaccine for taenia solium |
| CN112891523A (en) * | 2021-03-25 | 2021-06-04 | 遵义医科大学 | Preparation and identification method of taenia solium Ts14-3-3.3 DNA vaccine |
| CN112891523B (en) * | 2021-03-25 | 2023-07-04 | 遵义医科大学 | Preparation and identification method of taenia suis Ts14-3-3.3DNA vaccine |
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Application publication date: 20140312 |