CN101234196A - Coccidiosis recombination bacillus calmette-guerin vaccine and preparation - Google Patents
Coccidiosis recombination bacillus calmette-guerin vaccine and preparation Download PDFInfo
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- CN101234196A CN101234196A CNA2007100563707A CN200710056370A CN101234196A CN 101234196 A CN101234196 A CN 101234196A CN A2007100563707 A CNA2007100563707 A CN A2007100563707A CN 200710056370 A CN200710056370 A CN 200710056370A CN 101234196 A CN101234196 A CN 101234196A
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Abstract
The invention provides a coccidia recombinant BCG, which consists of a shuttling expression vector of coccidia recombinant BCG and an integration expression vector of coccidia recombinant BCG vaccine. A live vector vaccine of BCG has strong adjuvant effect on cellular immunity and humoral immunity and can express coccidian protein with high efficiency and lead the protein expressed to bring into play of immunity protection, thus reaching a goal of better chicken coccidia prevention with the advantages combined. The vaccine has great anti-coccodia effect, good thermal stability and is easy to transport and store; the immunity of the vaccine is long lasting, so that a single inoculation can continuously induce the immunity reaction to the 'target antigen', which can reduce the inoculating times, simplify the immune process and enhance the immunity effect towards the coccodia; the gene operation and the production process is simple and of high safety, so that the coccodia can express on the BCG cell wall continuously and stably; the product needs no purifying and can be directly used for immunity protection experiments, which omits the complicated processes of the post treatment of proteins, greatly reduces the cost and is convenient for popularizing.
Description
Technical field
The invention provides a kind of coccidiosis recombination bacillus calmette-guerin vaccine, comprise shuttle expression carrier coccidiosis recombination bacillus calmette-guerin vaccine and integrating expression vector coccidiosis recombination bacillus calmette-guerin vaccine vaccine, also disclose its preparation method simultaneously, belong to gene engineering technology field.
Background technology
Chicken coccidiosis is one of important diseases of the serious harm aviculture development that causes of a kind of unicellular parasitic protozoa by Eimeria, various places all over the world.Primary disease control for a long time mainly depends on medicine, but because the Drug resistance of chicken coccidiosis is on the rise, ubiquitous in addition drug residue, development new drug cost height, cycle are long, and immune effect is poor, problems such as immune programme for children complexity make people that sight has been turned to molecular vaccine.Booster immunization effect and the focus that the research of immunostimulant has been become current coccidial vaccine research.People once attempted the antigen gene of coccidiosis is expressed at different expression environment; but still there is not a kind of antigen gene that immunoprotection completely can be provided at present; and be subjected to the restriction of expression system, be that the recombinant protein antigen of prokaryotic expression system is relatively poor with escherichia coli.Therefore, be necessary to adopt a kind of efficient, cheap novel vaccine to chicken coccidiosis of gene engineering method development.
Bacillus calmette-guerin vaccine is most widely used in the world at present general, safest vaccine.Itself is a kind of good nonspecific immunoenhancer, can increase body's immunity, and the single inoculation can induce body to produce complete persistent cellular immunization and humoral immunization, and is easy to produce, and is cheap, is suitable for vast rural area.These advantages make bacillus calmette-guerin vaccine become a kind of very ideal live bacterial vaccines carrier of express recombinant exogenous gene.
Summary of the invention
The purpose of this invention is to provide a kind of coccidiosis recombination bacillus calmette-guerin vaccine, comprise the new generation vaccine of two kinds of resisting Eimeria tenellas, be i.e. shuttle expression carrier coccidiosis recombination bacillus calmette-guerin vaccine and integrating expression vector coccidiosis recombination bacillus calmette-guerin vaccine vaccine.
Technical solution of the present invention is as follows:
At first obtain the Eimeria tenella protective antigen gene; carry out the TA clone; order-checking identifies that correct back enzyme action reclaims the purpose fragment; link to each other with integrating expression vector PMV361 with the bacillus coli-mycobacteria shuttle expression carrier PMV261 that carries out endonuclease reaction equally respectively again; recombinant plasmid transformed is to BCG; obtain positive Eimeria tenella recombinant bacillus Calmette-Guerin vaccine through resistance and PCR screening, comprise shuttle expression carrier coccidiosis recombination bacillus calmette-guerin vaccine and integrating expression vector coccidiosis recombination bacillus calmette-guerin vaccine vaccine.
Two kinds of recombinant BCG vaccine bacterial strains provided by the present invention are with its called after rBCG PMV261-RHO and rBCG PMV361-RHO.
The present invention is an example with coccidiosis protective antigen Rhomboid gene, carries out the structure of shuttle expression carrier and integrating expression vector.
1, the preparation of shuttle expression carrier coccidiosis recombination bacillus calmette-guerin vaccine vaccine:
From the Eimeria tenella hybridization worm strain F2cDNA expression library that makes up, filter out 1 Rhomboid protein family related gene, open reading frame design primer according to the new gene order of Eimeria tenella Rhomboid of having cloned carries out PCR, the TA clone.Order-checking is carried out double digestion after identifying correctly, cutting glue reclaims, be connected with the pMV261 shuttle expression carrier that carries out same enzyme action, change among the DH5 α, recombiant plasmid Rho-261 electroporation is transformed in the bacillus calmette-guerin vaccine, and 37 ℃ are cultured to the logarithmic growth after date, screening BCG recon, PCR carries out 45 ℃ of thermal inductions expression after turning out to be positive colony, and gene expression product is carried out SDS-PAGE electrophoretic analysis and Western blotting evaluation.
The shuttle expression carrier coccidiosis recombination bacillus calmette-guerin vaccine vaccine rBCG PMV261-RHO for preparing is passed through collunarium eye dripping, oral, three kinds of different immunization route immunized chickses of cervical region subcutaneous injection with 100ug/ dosage only; and establish BCG simultaneously and organize and red, white matched group; three exempt from all backs oral vaccination Eimeria tenella egg capsule attacks the worm test, and indexs such as protective rate, the relative weight gain rate, OPG, ACI, humoral immunity level and cellular immune level detection are carried out overall merit.
Shuttle expression carrier coccidiosis recombination bacillus calmette-guerin vaccine vaccine rBCG PMV261-RHO can resist the attack of median dose coccidiosis; test group is compared with matched group, attacks behind the worm egg capsule output significantly to reduce, and body weight gain significantly increases; caecum lesion is less, and protective rate reaches more than 78.8%.Every index all obviously is better than matched group (the results are shown in Table 1).Wherein negative control group ACI value is 90.6, and rBCG PMV261-RHO immune group ACI value is respectively 208.1,181.4,167.2, illustrate and use the raising anticoccidial index that recombinant BCG vaccine can be in various degree, and collunarium eye dripping and oral group of ACI value all reach more than 180, and be very effective with these two kinds of approach immunity anticoccidial effects.Three groups of the BCG immunity its ACI values are between 154.6 ~ 171.0, and effect points out us to use BCG to strengthening chicken immunity to coccidiosis protection certain effect to be arranged also separately also apparently higher than matched group, and is wherein better with the oral way immune effect.Effective cellular immunization and humoral immunization be can induce behind the shuttle expression carrier coccidiosis recombination bacillus calmette-guerin vaccine vaccine immunity chickling, CD4+, CD8+T cell quantity and specific antibody titre shown as apparently higher than matched group (P＜0.01).
2, the preparation of integrating expression vector coccidiosis recombination bacillus calmette-guerin vaccine vaccine:
Open reading frame according to the new gene order of Eimeria tenella Rhomboid of having cloned designs the design of primers primer and introduces restriction enzyme site, carry out PCR, the TA clone, order-checking is carried out double digestion after identifying correctly, cutting glue reclaims, be connected with the pMV361 integrating expression vector that carries out same enzyme action, change among the DH5 α, recombiant plasmid Rho-361 electroporation is transformed in the bacillus calmette-guerin vaccine, 37 ℃ are cultured to the logarithmic growth after date, screening BCG recon, PCR carry out 45 ℃ of thermal inductions expression after turning out to be positive colony, and gene expression product is carried out SDS-PAGE electrophoretic analysis and Western blotting evaluation.
Recombinant bacillus Calmette-Guerin vaccine rBCG PMV361-RHO is passed through collunarium eye dripping, oral, three kinds of different immunization route immunized chickses of cervical region subcutaneous injection with 100ug/ dosage only; carry out the negative control experiment simultaneously; three exempt from all backs oral vaccination Eimeria tenella egg capsule attacks the worm test, and indexs such as protective rate, the relative weight gain rate, OPG, ACI, humoral immunity level and cellular immune level detection are carried out overall merit.
Use the raising anticoccidial index that recombinant BCG vaccine can be in various degree, wherein rBCGPMV361-RHO collunarium eye dripping and oral immunity group ACI value reach 188.2 and 187.8, have good anticoccidial effect.Integrating expression vector coccidiosis recombination bacillus calmette-guerin vaccine vaccine rBCG PMV361-RHO can resist the attack of median dose coccidiosis; test group is compared with matched group; attack behind the worm egg capsule output and significantly reduce; body weight gain significantly increases; caecum lesion is less, and three kinds of immunization route protective rates are respectively 80%, 70.6%, 63.5% (the results are shown in Table 2).More obvious with collunarium eye dripping immunization ways effect.With inducing effective cellular immunization and humoral immunization behind the integrating expression vector coccidiosis recombination bacillus calmette-guerin vaccine vaccine immunity chickling, CD4+, CD8+T cell quantity and specific antibody titre all have in various degree raising (P＜0.05) than negative control group.
With two kinds of coccidiosis recombination bacillus calmette-guerin vaccines provided by the invention by collunarium eye dripping, oral, three kinds of different immunization route immunized chickses of cervical region subcutaneous injection after; oral vaccination Eimeria tenella egg capsule is attacked the worm test, carries out overall merit by indexs such as protective rate, the relative weight gain rate, OPG, ACI, humoral immunity level and cellular immune level detections.The result shows, coccidiosis recombination bacillus calmette-guerin vaccine rBCG PMV261-RHO and rBCGPMV361-RHO group all have tangible immunologic enhancement, each index all is better than negative control group, can resist the attack of Eimeria tenella egg capsule effectively, the ACI value arrives more than 180, has good anticoccidial effect.
Good effect of the present invention is: the bacillus calmette-guerin vaccine live vector vaccine itself has stronger cellular immunization and humoral immunization adjuvant effect; and can efficiently express coccidiosis albumen again; make expressed proteins bring into play good immanoprotection action; both advantages make up, and reach the purpose of better prevention chicken coccidiosis.And this new generation vaccine Heat stability is good transports and preserves more or less freely, is easy to produce, and product does not need purification, can be directly used in the immunoprotection test and remove the complicated procedures of forming of protein post processing from, thereby greatly reduce cost, is suitable for vast rural area.The used Rhomboid gene of the present invention is the new gene of an Eimeria tenella obtaining of my laboratory.Rhomboid albumen is the participation epidermal growth factor of discovered in recent years and the actuator of EGF-R ELISA signal transduction process, and found that Rhomboid sticks with apical organ door protozoacide and to invade host cell relevant, the value of Rhomboid gene as the vaccine candidate gene is described.
Such live vector vaccine can be brought into play the stronger cellular immunization adjuvant effect of bacillus calmette-guerin vaccine itself, can make expressed proteins performance immanoprotection action again, reaches the purpose of better prevention chicken coccidiosis.Therefore the development of these two kinds of vaccines has boundless prospect and using value.
Description of drawings
Fig. 1 is a PMV261-Rho vector construction sketch map.
Fig. 2 is a PMV361-Rho vector construction sketch map.
The specific embodiment
The present invention is an example with coccidiosis protective antigen Rhomboid gene, carries out the structure of shuttle expression carrier and integrating expression vector, does not limit the present invention in any form.
The preparation of coccidiosis recombination bacillus calmette-guerin vaccine vaccine of the present invention
Embodiment 1
The preparation process of shuttle expression carrier coccidiosis recombination bacillus calmette-guerin vaccine vaccine:
Design two pairs of primers and introduce restriction enzyme site with reference to Rhomboid gene DNA sequence and shuttle vector pMV261 physical map.Forward primer QF1:5`-CTGACTGCAGATGTC GGACA TCGAA TCCCAGAG-3`; Wherein the 5` end contains the PstI site; Downstream primer QR:5`-GACT ATCGAT TTATG CGCATCCCAT GGGCA AAGG-3`; The 5` end contains the ClaI site.The PCR purified product is cloned into go forward side by side performing PCR, enzyme action and the order-checking of PMD18-T carrier to be identified, gel reclaims fragment and is connected with the shuttle expression carrier pMV261 that carries out enzyme action equally, screen recombiant plasmid after connecting product Transformed E .coli DH5 α competent cell, carry out double digestion reaction, recombiant plasmid called after PMV261-Rho (as shown in Figure 1) with Pst I, Cla I.BCG is inoculated in the MB7H9 ADC fluid medium, and 37 ℃ are cultured to exponential phase, centrifugal collection thalline, and precipitation, is used for electricity and transforms in last resuspended 1ml 10% glycerol with the washing of 10% glycerol.Get 60-80ulBCG competence bacteria liquid adding 0.1ug recombiant plasmid PMV261-Rho and place electric revolving cup.Electricity is worn parameter: voltage 2.5KV, electric capacity 25 μ F, resistance 1000 Ω.Electroporation adds in the MB7H9 ADC culture medium after transforming immediately, coats behind 37 ℃ of cultivation 2d to contain 20ug/mlKan MB7H9 ADC culture medium flat plate, grows the conversion bacterium colony behind about 3w.Picking BCG recon from the culture medium flat plate is inoculated in fluid medium (containing Kan), and 37 ℃ are cultured to exponential phase, PCR induces 4h in 45 ℃ of water-baths, centrifugal collection thalline after confirming positive colony, and handle, add equal-volume 2 * SDS-PAGE sample-loading buffer at last, 100 ℃ of 5min.Getting the above-mentioned bacterium liquid of 12ul SDS-PAGE analysis and Western blotting identifies.
Embodiment 2
The preparation process of integrating expression vector coccidiosis recombination bacillus calmette-guerin vaccine vaccine:
Design two pairs of primers and introduce restriction enzyme site with reference to Rhomboid gene DNA sequence and shuttle vector pMV361 physical map.Forward primer QF2:CTGA CAGCTG ATGTCG GACATC GAATCC CAGAG; Wherein the 5` end contains Pvu II site; Downstream primer QR:5`-GACT ATCGAT TTATG CGCAT CCCATGGGCA AAGG-3`; The 5` end contains the ClaI site.The PCR purified product is cloned into go forward side by side performing PCR, enzyme action and the order-checking of PMD18-T carrier to be identified, gel reclaims segment and is connected with integrating expression vector pMV361, screen recombiant plasmid after connecting product Transformed E .coli DH5 α competent cell, with carrying out double digestion reaction, recombiant plasmid called after PMV361-Rho (as shown in Figure 2) with Pvu II, Cla I.Get 60-80ul competence BCG bacterium liquid adding 0.1ug recombiant plasmid PMV361-Rho and place electric revolving cup.Electricity is worn parameter: voltage 2.5KV, electric capacity 25 μ F, resistance 1000 Ω.Electroporation adds in the MB7H9 ADC culture medium after transforming immediately, coats MB7H9 ADC culture medium flat plate behind 37 ℃ of cultivation 2d.Picking BCG recon from the culture medium flat plate is inoculated in fluid medium, and 37 ℃ are cultured to exponential phase, induce 4h in 45 ℃ of water-baths behind the PCR confirmation positive colony, centrifugal collection thalline, and handle, add equal-volume 2 * SDS-PAGE sample-loading buffer at last, 100 ℃ of 5min.Getting the above-mentioned bacterium liquid of 12ul SDS-PAGE analysis and Western blotting identifies.
Test example 1
The purposes of shuttle expression carrier coccidiosis recombination bacillus calmette-guerin vaccine vaccine
Laboratory animal adopts the sea that has just gone out shell blue little public young, during chickling 6 ages in days, is divided into 8 groups at random, 20 every group.During 7 ages in days shuttle expression carrier coccidiosis recombination bacillus calmette-guerin vaccine vaccine rBCG PMV261-RHO is passed through collunarium eye dripping, oral, three kinds of different immunization route immunized chickses of cervical region subcutaneous injection with 100ug/ dosage only, and establish BCG collunarium eye dripping immune group, BCG oral immunity group, BCG cervical region subcutaneous injection immune group and red white matched group simultaneously.Red white matched group inoculation PBS, red matched group is attacked worm at last, and white matched group is not attacked worm.Divide three immunity, in each week at interval, three exempt from all backs oral vaccination Eimeria tenella egg capsule 1 * 10
4Individual/as only to attack the worm test, carry out the following index and judge, comprise protective rate, the relative weight gain rate, OPG, ACI etc., the blood sampling of immunity back, flow cytometer is to CD4
+, CD8
+Carry out the cellular level immune detection, separation of serum ELISA method detects humoral immunity level.
Attack before the worm every group of chicken and get 10 at random, attack behind the worm every other day chicken to correspondence, observe the body weight change situation of attacking chicken behind the worm by only weighing by only weighing respectively and label record, and last weightening finish situation, the relative weight gain calculated; Check feces every day after attacking worm, and collected respectively since the 5th day and respectively to organize feces, behind the mixing, gets 1g for every group, adds the 10mL tap water and make 10 times of diluents, gets one and place blood cell counting plate, numeration coccidian oocyst sum under low power lens; After attacking worm the 7th day, get 5 chickens for every group and cut open and kill, observe caecum lesion, keep the score by the lesion score method of Johnson design.Calculating ACI=the relative weight gain rate+survival rate-pathological changes value-egg capsule keeps the score.
The result shows that every index all obviously is better than the not immune worm matched group of attacking.Illustrate that the immune programme for children that we adopt is safely and effectively, the ACI of negative control group only is 90.6, and use the raising anticoccidial index that BCG can be different separately, wherein oral group of ACI of BCG reached 171.0, brought into play the effect of BCG as immunostimulant, that effect is particularly outstanding is shuttle expression carrier coccidiosis recombination bacillus calmette-guerin vaccine vaccine rBCG PMV261-RHO provided by the present invention, immunization ways adopts collunarium eye dripping and oral, anticoccidial Index A CI value all reaches more than 180, illustrates that anticoccidial effect is very effective.Shuttle expression carrier coccidiosis recombination bacillus calmette-guerin vaccine vaccine rBCG PMV261-RHO can resist the attack of median dose coccidiosis; test group is compared with matched group, attacks behind the worm egg capsule output significantly to reduce, and body weight gain significantly increases; caecum lesion is less, and protective rate reaches more than 78.8% and (the results are shown in Table 1).
Behind the new generation vaccine rBCG PMV261-RHO immunized chicks with invention,, get 10 chicken sacrificed by exsanguination at random for every group, get spleen, add 2mL PBS, on 200 mesh sieves, grind, be diluted to cell suspension (10 with PBS in 1 week of back of immunity for the third time
7Individual/mL).Get 100 μ L cell suspension, add the anti-CD4 of FITC labelling
+Anti-CD8 with the PE labelling
+The anti-chicken monoclonal antibody of rabbit, lucifuge effect 40min washes 2 times with the PBS washing liquid then, adds 0.5mL fluorescence and preserves liquid, detects CD4 with flow cytometer
+, CD8
+The lymphocytic quantity of T, and the gained data are carried out statistical analysis with SPSS software.The result shows immune group and BCG group CD4
+, CD8
+Variable is apparently higher than negative control group, and the CD4+ of rBCG PMV261-RHO collunarium group chicken, CD8+T lymphocyte number average raise, and compares difference all extremely significantly (P＜0.01) with other groups.Heart blood sampling before each immunity, separation of serum, detects IgG situation of change in the chicken serum by indirect ELISA method as envelope antigen with Eimeria tenella F2 strain sporozoite protein.One exempt from each experimental group of back and compare the IgG titre with matched group and change not quite as a result, difference is not remarkable.Two exempt from back IgG titre rises gradually, after the immunity, each experimental group all shows certain antibody titer, wherein for the third time, oral group of serum antibody absorbance of rBCG PMV261-RHO collunarium group and rBCG PMV261-RHO is the highest, compares difference extremely significantly (P＜0.01) in other group.
Test example 2
The purposes of integrating expression vector coccidiosis recombination bacillus calmette-guerin vaccine vaccine
Laboratory animal adopts the sea that has just gone out shell blue little public young, during chickling 6 ages in days, is divided into 8 groups at random, 20 every group.During 7 ages in days integrating expression vector coccidiosis recombination bacillus calmette-guerin vaccine vaccine rBCG PMV361-RHO is passed through collunarium eye dripping, oral, three kinds of different immunization route immunized chickses of cervical region subcutaneous injection with 100ug/ dosage only, and establish BCG collunarium eye dripping immune group, BCG oral immunity group, BCG cervical region subcutaneous injection immune group and red white matched group simultaneously.Red white matched group inoculation PBS, red matched group is attacked worm at last, and white matched group is not attacked worm.Divide three immunity, in each week at interval, three exempt from all backs oral vaccination Eimeria tenella egg capsule 1 * 10
4Individual/as only to attack the worm test, carry out the following index and judge, comprise protective rate, the relative weight gain rate, OPG, ACI etc., the blood sampling of immunity back, flow cytometer is to CD4
+, CD8
+Carry out the cellular level immune detection, separation of serum ELISA method detects humoral immunity level.
Attack before the worm every group of chicken and get 10 at random, attack behind the worm every other day chicken to correspondence, observe the body weight change situation of attacking chicken behind the worm by only weighing by only weighing respectively and label record, and last weightening finish situation, the relative weight gain calculated; Check feces every day after attacking worm, and collected respectively since the 5th day and respectively to organize feces, behind the mixing, gets 1g for every group, adds the 10mL tap water and make 10 times of diluents, gets one and place blood cell counting plate, numeration coccidian oocyst sum under low power lens; After attacking worm the 7th day, get 5 chickens for every group and cut open and kill, observe caecum lesion, keep the score by the lesion score method of Johnson design.Calculating ACI=the relative weight gain rate+survival rate-pathological changes value-egg capsule keeps the score.
Use the raising anticoccidial index that recombinant BCG vaccine can be in various degree, wherein rBCGPMV361-RHO collunarium eye dripping and oral immunity group ACI value reach 188.2 and 187.8, have good anticoccidial effect.Integrating expression vector coccidiosis recombination bacillus calmette-guerin vaccine vaccine rBCG PMV361-RHO can resist the attack of median dose coccidiosis; test group is compared with matched group; attack behind the worm egg capsule output and significantly reduce; body weight gain significantly increases; caecum lesion is less, and three kinds of immunization route protective rates are respectively 80%, 70.6%, 63.5% (the results are shown in Table 2).More obvious with collunarium eye dripping immunization ways effect.
Behind the new generation vaccine rBCG PMV361-RHO immunized chicks with invention,, get 10 chicken sacrificed by exsanguination at random for every group, get spleen, be diluted to cell suspension, get 100 μ L cell suspension, add the anti-CD4 of FITC labelling with PBS in 1 week of back of immunity for the third time
+Anti-CD8 with the PE labelling
+The anti-chicken monoclonal antibody of rabbit detects CD4 with flow cytometer
+, CD8
+The lymphocytic quantity of T, and the gained data are carried out statistical analysis with SPSS software.The result shows immune group and BCG group CD4
+, CD8
+Variable is apparently higher than negative control group.Heart blood sampling before each immunity, separation of serum, detects IgG situation of change in the chicken serum by indirect ELISA method as envelope antigen with Eimeria tenella F2 strain sporozoite protein.One exempt from each experimental group of back and compare the IgG titre with matched group and change not quite as a result, difference is not remarkable.Two exempt from back IgG titre rises gradually, and after the immunity, each experimental group all shows certain antibody titer for the third time.
The protection effect that table 1 shuttle vector recombinant bacillus Calmette-Guerin vaccine rBCG PMV261-RHO attacks E.tenella
| OPG value * 10 6Individual | Weightening finish (g) | Caecum lesion is kept the score | ACI | ||||
| Grouping | Meansigma methods | Protective rate | Meansigma methods | The relative weight gain | Meansigma methods | Relative pathological changes | ACI |
| Oral group of rBCG PMV261-RHO collunarium group rBCG PMV261-RHO | 0.275 0.3 | 87.1％ 85.9％ | 93.5 74.9 | 121.6％ 97.4％ | 1.75 1.9 | 51.4％ 62.9％ | 208.1 181.4 |
| RBcG PMV261-RHO cervical region subcutaneous injection group | 0.45 | 78.8％ | 66.68 | 86.7％ | 2.1 | 71.4％ | 167.2 |
| The white matched group of oral group of BCG cervical region of the BCG collunarium group BCG red matched group of subcutaneous injection group | 0.675 0.875 1.125 2.125 | 68.2％ 58.8％ 47.1％ 0 | 62.6 75.4 64.3 43.9 76.9 | 81.4％ 98.1％ 83.6％ 57.1％ 100％ | 2.5 2.78 2.8 3.5 | 71.4％ 79.4％ 80％ 100％ | 156.9 171.0 154.6 90.6 200.0 |
The protection effect that table 2 integration vector recombinant bacillus Calmette-Guerin vaccine rBCG PMV361-RHO attacks E.tenella
| 0PG value * 10 6Individual | Weightening finish (g) | Caecum lesion is kept the score | ACI | ||||
| Grouping | Meansigma methods | Protective rate | Meansigma methods | The relative weight gain | Meansigma methods | Relative pathological changes | ACI |
| Oral group of rBCG PMV361-RHO collunarium group rBCG PMV361-RHO | 0.425 0.625 | 80％ 70.6％ | 81.7 84.46 | 106.2％ 110.0％ | 1.8 2.2 | 50％ 54.3％ | 188.2 187.8 |
| RBcG PMV361-RHO cervical region subcutaneous injection group | 0.775 | 63.5％ | 79.2 | 103.0％ | 2.5 | 60％ | 152.7 |
| The white matched group of oral group of BCG cervical region of the BCG collunarium group BCG red matched group of subcutaneous injection group | 0.675 0.875 1.125 2.125 | 68.2％ 58.8％ 47.1％ 0 | 62.6 75.4 64.3 43.9 76.9 | 81.4％ 98.1％ 83.6％ 57.1％ 100％ | 2.5 2.78 2.8 3.5 | 711.4％ 79.4％ 80％ 100％ | 156.9 171.0 154.6 90.6 200.0 |
Claims (3)
1, a kind of coccidiosis recombination bacillus calmette-guerin vaccine; it is characterized in that: at first obtain the coccidiosis protective antigen gene; carry out the TA clone; order-checking identifies that correct back enzyme action reclaims the purpose fragment; link to each other with bacillus coli-mycobacteria shuttle expression carrier PMV261 that carries out endonuclease reaction equally or integrating expression vector PMV361 respectively again; recombinant plasmid transformed obtains positive coccidiosis recombination bacillus calmette-guerin vaccine to BCG through resistance and PCR screening.
2, a kind of shuttle expression carrier coccidiosis recombination bacillus calmette-guerin vaccine vaccine, prepare by following steps: from make up coccidiosis hybridization worm strain F2cDNA expression library, filter out 1 Rhomboid protein family related gene, open reading frame design primer according to the new gene order of Eimeria tenella Rhomboid of having cloned carries out PCR, the TA clone; Order-checking is carried out double digestion after identifying correctly, cutting glue reclaims, be connected with the pMV261 shuttle expression carrier that carries out same enzyme action, change among the DH5 α, recombiant plasmid Rho-261 electroporation is transformed in the bacillus calmette-guerin vaccine, and 37 ℃ are cultured to the logarithmic growth after date, screening BCG recon, PCR carries out 45 ℃ of thermal inductions expression after turning out to be positive colony, and gene expression product is carried out SDS-PAGE electrophoretic analysis and Western blotting evaluation.
3, a kind of integrating expression vector coccidiosis recombination bacillus calmette-guerin vaccine vaccine, prepare by following steps: the open reading frame according to the new gene order of coccidiosis Rhomboid of having cloned designs the design of primers primer and introduces restriction enzyme site, carry out PCR, the TA clone, order-checking is carried out double digestion after identifying correctly, cutting glue reclaims, be connected with the pMV361 integrating expression vector that carries out same enzyme action, change among the DH5 α, recombiant plasmid Rho-361 electroporation is transformed in the bacillus calmette-guerin vaccine, 37 ℃ are cultured to the logarithmic growth after date, screening BCG recon, PCR carries out 45 ℃ of thermal inductions expression after turning out to be positive colony, and gene expression product is carried out SDS-PAGE electrophoretic analysis and Western blotting evaluation.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US9603915B2 (en) | 2013-02-14 | 2017-03-28 | The Board of Trustees of the University of Akansas | Compositions and methods of enhancing immune responses to Eimeria or limiting Eimeria infection |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9603915B2 (en) | 2013-02-14 | 2017-03-28 | The Board of Trustees of the University of Akansas | Compositions and methods of enhancing immune responses to Eimeria or limiting Eimeria infection |
| US9884099B2 (en) | 2013-02-14 | 2018-02-06 | The Board Of Trustees Of The University Of Arkansas | Compositions and methods of enhancing immune responses to Eimeria or limiting Eimeria infection |
| US10328137B2 (en) | 2013-02-14 | 2019-06-25 | The Board Of Trustees Of The University Of Arkansas | Compositions and methods of enhancing immune responses to Eimeria or limiting Eimeria infection |
| US10792351B2 (en) | 2013-02-14 | 2020-10-06 | The Board Of Trustees Of The University Of Arkansas | Compositions and methods of enhancing immune responses to Eimeria or limiting Eimeria infection |
| US11364290B2 (en) | 2013-02-14 | 2022-06-21 | The Board Of Trustees Of The University Of Arkansas | Compositions and methods of enhancing immune responses to eimeria or limiting eimeria infection |
| US11904005B2 (en) | 2013-02-14 | 2024-02-20 | The Board Of Trustees Of The University Of Arkansas | Compositions and methods of enhancing immune responses to Eimeria or limiting Eimeria infection |
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