CN102352338A - Brucella shell as well as preparation method and application thereof - Google Patents
Brucella shell as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the field of microorganism, and in particular discloses a Brucella shell as well as a preparation method and application thereof. The preparation method comprises the following steps: cloning a lysis gene E having a nucleotide sequence shown in SEQ ID NO:1 to a plasmid pBV220 so as to obtain a recombinant plasmid pBV220::E; subsequently, amplifying the recombinant plasmid pBV220::E, and connecting the amplifying product to a plasmid pBBR1MCS-2; then transforming to Brucella; carrying out propagation culture on the recombinant strain at the temperature of 25-30 DEG C until the OD600 value of the strain is 0.8-1.2; raising the temperature to 37-45 DEG C until the OD600 value is stable; then centrifuging and collecting strain; and washing and then carrying out freeze thawing with a hypertonic solution so as to obtain the Brucella shell. According to the invention, the Brucella shell with high safety and good protection effect is prepared by transforming a fusion sequence of a temperature-controlling regulation sequence and the lysis gene E to the Brucella through adopting biotechnology, and the obtained Brucella shell has an important significance in control of epidemic and propagation of Brucella disease.
Description
Technical field
The present invention relates to microorganism field, relate to a kind of brucella bacterium shell and its production and application in particular.
Background technology
Brucella (Brucella spp.) is a kind of born of the same parents' endoparasitism, the former bacterium of Amphixenosis, and brucellosis (cloth sick) be meant by brucella cause a kind of popular extensively, infectivity is strong, very harmful, the people beast that is difficult to effect a radical cure after infecting suffers from transmissible disease altogether.The sick cardinal symptom of animal cloth is heating, miscarriage, sterile, chronic arthritis and nervous lesion etc., can cause that conceived jenny miscarries in later stage of pregnancy, and testitis of buck and epididymitis etc.; People's cloth is sick main through mucocutaneous, digestive tube and respiratory tract infection, and its clinical manifestation be to generate heat for a long time, with hidrosis, arthrodynia, hepatosplenomegaly and reproductive system damage etc.After the people infects brucella, need long antibiotic therapy, and tend to stay more serious sequela.
The domestic animal that infects is the sick main contagium of people's cloth, and the people is mainly through contacting ill livestock and products thereof or its pollutent infects the cloth disease.Cloth disease not only serious harm human and animal's health, influences livestock industry, tourism, international trade and Economic development simultaneously.This disease all is widely current all over the world, and the annual financial loss that therefore causes is up to multi-million dollar.The cloth disease is wide in the popular scope of China, harm is serious, after new China sets up, carried out comprehensively and the control of system, has controlled the sick generation of cloth and popular effectively.Yet since the nineties in last century, the sick sickness rate of Chinese people and animal cloth has the trend of obvious rising again.
Mostly the brucella vaccine of current use is attenuated vaccine, though this type vaccine has certain immune protective effect, also has very significant disadvantages, and promptly toxicity is big, and atavism is serious, can damage people and animal body, even morbidity.Therefore developing a kind of brucella vaccine with good protection effect and security has important and practical meanings.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of brucella bacterium shell and its production and application, make said brucella bacterium shell have good security and protection effect, be better than existing attenuated vaccine.
For realizing above-mentioned purpose, the present invention provides following technical scheme:
A kind of preparation method of brucella bacterium shell comprises:
(Bacterial ghosts BG) is a kind of novel bacteria vaccine to the bacterium shell, is normally formed at bacterium surface by the crack protein E of PhiX174 phage and strides fenestra shape structure.Hydrophobic protein of forming by 91 amino acid of the E genes encoding of phage PhiX174; This albumen can play the effect that connects the inside and outside film of bacterium through oligomerization; Thereby form a kind of poroid passage; The diameter of poroid passage is between 40-200nm; Its size is different because of the bacterium individual difference, relates generally to the variation that cytolemma took place in size cases and the cracking process of sieve aperture on degree that the bacterium self-dissolving takes place, the peptidoglycan layer.In a single day poroid passage forms, and under the effect of high osmotic pressure, bacterium content passing hole channel is discharged in cell, thereby forms a bacterium ghost that does not have composition in nucleic acid, rrna and other born of the same parents.
The bacterium shell is to be prepared from non-denaturation method; Bacterium thalline and various antigenic structure have been kept intactly; Natural adventitia of bacterium and self-faced antigen have been kept; The structure such as lipopolysaccharides and peptidoglycan that high immunostimulating is arranged on the adventitia; The effective constituent of these cell surfaces can be discerned by scavenger cell or antigen presenting cell, and then excites effective immunoreaction process.In addition, self also has adjuvanticity the bacterium shell, after transforming, can be used as vaccine carrier.
Therefore; The present invention adopts suitable primer, plasmid etc., prepares brucella bacterium shell through biotechnology means such as clone, reorganization, conversions, because brucella inner cell content has leaked; Belong to inactivated vaccine, so its security is apparently higher than existing attenuated vaccine.
Wherein, brucella according to the invention is preferably brucella S2 bacterial strain (being pig kind S2 strain), brucella M5 bacterial strain (being sheep kind M5 strain), brucella RB51 bacterial strain (being ox kind RB51 strain) or brucella 104M bacterial strain (being human 104M strain).
In preparation method according to the invention; Plasmid pBV220 is the commercial goods plasmid; Have temperature control on it and regulate sequence λ pL/pR-cI857 and definite multiple clone site; The present invention will be cloned into the downstream that sequence λ pL/pR-cI857 is regulated in temperature control by the lysis genes E of nucleotide sequence through directed cloning shown in SEQ ID NO:1; Obtain recombinant plasmid pBV220::E, so that in the preparation process of follow-up brucella bacterium shell, control the expression of said lysis genes E through controlled temperature.
Wherein, the said lysis genes E of step 1 is prepared by following method:
Use the forward primer LE-F of nucleotide sequence shown in SEQ ID NO:4 and shown in SEQ ID NO:5 the reverse primer LE-R of nucleotide sequence from phage PhiX174, increase and obtain lysis genes E.
Because plasmid pBV220 can not duplicate in brucella; So the present invention uses the forward primer BRU1 of nucleotide sequence shown in SEQ ID NO:2 and the λ pL/pR-cI857 among the reverse primer BRU2 amplification recombinant plasmid pBV220::E of nucleotide sequence and the fusion sequence of lysis genes E shown in SEQ ID NO:3; And the product after will increasing is connected among the broad host range plasmid carrier pBBR1MCS-2 between the SpeI and SalI restriction enzyme site; Recombinant plasmid pBBR1MCS-2-E is transformed in the brucella bacterial strain, and then allows lysis genes E in brucella, express through temperature control.
Plasmid pBBR1MCS-2 is so kind as to give by upright University Medical Center professor Kovach of Louisiana, United States, and the pBBR1MCS-2 collection of illustrative plates is referring to Fig. 1, and sequence is seen SEQ ID NO:6.
In order to collect brucella bacterium shell well, the present invention breeds cultivation with recombinant bacterial strain at 25-30 ℃ earlier, because the existence of temperature control sequence is arranged, can not express at lysis genes E below 30 ℃, guarantees that the brucella bacterial strain can breed its OD
600Value is preferably 1.0 for 0.8-1.2, makes biomass reach suitable degree, and the cracking after too high biomass is prone to make it is insufficient, causes the existence of a large amount of viable bacterias, influences security; And low excessively biomass is not easy to collect the bacterium shell after enough cracking.
After breeding is cultivated, be warming up to 37-45 ℃ rapidly and carry out inducing culture to its OD
600Value stabilization, temperature control system is in the expression that just can not check lysis genes E more than 37 ℃, and therefore a large amount of crack protein E is expressed, and the brucella bacterial strain is carried out cracking, forms the bacterium shell, the OD of brucella bacterial strain in cracking process
600Value is constantly to descend, and when its value stabilization, accomplishes cracking, and the cracking time is different because of different brucella bacterial strains, generally about 2 days.
After accomplishing cracking; Common brucella bacterial strain more than 99.9% all can cracking form the bacterium shell; But because the restriction of prior art is difficult to reach 100% cracking; Therefore in order further to guarantee the security of bacterium shell; The present invention is with the bacterium shell; Be that the hypertonic solution freeze thawing is used in centrifugal thalline washing back of collecting, guarantee the viable bacteria that deactivation is remaining.
In preparation method according to the invention, the said conversion of step 3 is preferably electricity and transforms, and said breeding culture temperature is 28 ℃, and said inducing culture temperature is 42 ℃, and said hypertonic solution is 5% sodium chloride solution.
In addition, the present invention also provides a kind of brucella bacterium shell and the application of said brucella bacterium shell in the vaccine of preparation prevention brucellosis by preparing method's preparation according to the invention.
Vaccine according to the invention comprises the said brucella bacterium shell and the pharmaceutically acceptable carrier of significant quantity.Wherein, medically acceptable carrier is the common carrier that is used to prepare vaccine well known in the art, also can comprise immunostimulant or immunomodulator, for example known medicament such as Freund's complete adjuvant and Freund's incomplete adjuvant.
Brucella bacterium shell by preparing method's preparation according to the invention through morphological observation, has typical bacterium shell characteristic, has effectively kept its outer membrane structure, and most intracellular organic matter is released.The security and the immunity test that carry out through BALB/c mouse confirm; Brucella bacterium shell according to the invention side effect is little, safe; But inducing mouse produces protection antibody; Compare with attenuated live vaccines; Can produce stronger immunological memory, can protect mouse to avoid the brucellar attack of strong dosage.
Can know by above technical scheme; The fusion sequence that the present invention adopts the biotechnology means that sequence λ pL/pR-cI857 and lysis genes E are regulated in temperature control is transformed in the brucella; Be prepared into the bacterium shell that side effect is little, safe, protection is effective; Popular and propagation to the control brucellosis is significant, has a extensive future.
Description of drawings
Shown in Figure 1 is the pBBR1MCS-2 plasmid map;
Shown in Figure 2 for recombinant plasmid pBBR1MCS-2-E carries out SpeI/SalI double digestion checking electrophorogram, wherein swimming lane 1 and 2 is that recombinant plasmid pBBR1MCS-2-E carries out the evaluation of SpeI/SalI double digestion, and swimming lane 3 is λ-Hind III degest dna molecular amount standards;
Shown in Figure 3 is that embodiment 1 recombinant bacterial strain PCR identifies electrophorogram, and wherein swimming lane 1 is the DL2000DNA molecular weight standard, and swimming lane 2 is the recombinant bacterial strain amplification;
Shown in Figure 4 is embodiment 1 brucella bacterium shell electromicroscopic photograph;
Shown in Figure 5 is embodiment 1 brucella bacterium shell electromicroscopic photograph;
Shown in Figure 6 is brucella S2 bacterial strain electromicroscopic photograph;
Shown in Figure 7 is brucella S2 bacterial strain electromicroscopic photograph;
Shown in Figure 8 is that the BALB/c mouse mean body weight changes broken line graph in the safety testing of the present invention; Wherein ordinate zou is a body weight (unit: g); X-coordinate is a time (unit: day); Broken line 1 is the broken line (the 1st group of broken line) of injection brucella S2 bacterial strain attenuated live vaccines group; Broken line 2 is the brucella bacterium shell group (the 2nd group of broken line) of injection embodiment 1 preparation, and broken line 3 is the negative control group (the 3rd group of broken line) of injecting normal saline;
Shown in Figure 9 is that serum IgG antibody detects column diagram after the embodiment 1 brucella bacterium shell immunity; Wherein X-coordinate is an immunity back time (unit: week); Ordinate zou is the antibody titers logarithm; Cylindricality 1 is the column diagram (the 2nd group of column diagram) of the brucella bacterium shell of injection inoculation embodiment 1 preparation, the column diagram (the 1st group of column diagram) of injection brucella S2 bacterial strain attenuated live vaccines group.
Embodiment
The invention discloses a kind of brucella bacterium shell and its production and application, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as and are included in the present invention.Preparing method according to the invention, product are described through preferred embodiment; The related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use technology of the present invention.
Below in conjunction with embodiment, further set forth the present invention.
Embodiment 1: prepare brucella bacterium shell according to the invention
Use the forward primer LE-F of nucleotide sequence shown in SEQ ID NO:4 and shown in SEQ ID NO:5 the reverse primer LE-R of nucleotide sequence from phage PhiX174 (available from MBI company), increase and obtain the lysis genes E of nucleotide sequence shown in SEQ ID NO:1;
To shown in SEQ ID NO:1, be cloned among the plasmid pBV220 (giving birth to worker's biotechnology company limited) by the lysis genes E of nucleotide sequence, obtain recombinant plasmid pBV220::E available from Shanghai;
Use the forward primer BRU1 of nucleotide sequence shown in SEQ ID NO:2 and the reverse primer BRU2 amplification recombinant plasmid pBV220::E of nucleotide sequence shown in SEQ ID NO:3; Product after the amplification is connected among the plasmid pBBR1MCS-2 between the SpeI and SalI restriction enzyme site, obtains recombinant plasmid pBBR1MCS-2-E;
Recombinant plasmid pBBR1MCS-2-E is transformed in the brucella S2 bacterial strain and obtains recombinant bacterial strain, recombinant bacterial strain is bred at 28 ℃ be cultured to its OD
600Value is to be warming up to 42 ℃ rapidly after 1.0 to carry out inducing culture to its OD
600Value stabilization, centrifugal then collection thalline, the washing back promptly gets with 5% sodium-chlor freeze thawing.
Embodiment 2: the checking of recombinant plasmid pBBR1MCS-2-E
The recombinant plasmid pBBR1MCS-2-E that obtains among the embodiment 1 is carried out the checking of SpeI/SalI double digestion, and the result sees Fig. 2.Can be known that by Fig. 2 it is consistent with expection that enzyme is cut qualification result, it is correct to confirm that recombinant plasmid pBBR1MCS-2-E makes up.
Embodiment 3: the checking of recombinant bacterial strain
Carry out the PCR checking with obtaining recombinant bacterial strain among the embodiment 1, see Fig. 3.Fig. 3 fusion fragment that sequence λ pL/pR-cI857 and lysis genes E are regulated in clone's temperature control that confirmed from recombinant bacterial strain, to have increased shows that recombinant bacterial strain makes up successfully.
Embodiment 4: brucellar cleavage rate statistics according to the invention
After the bacterium liquid of (back is accomplished in cracking) behind (breeding cultivate accomplish back) before embodiment 1 inducing culture and the inducing culture diluted with suitable multiple respectively, through colony counting method mensuration viable count (CFU/mL), the lysis efficiency of calculating recombinant bacterial strain.Triplicate is averaged, and the result shows that lysis efficiency reaches more than 99.9%.
Embodiment 5: the electron microscopic observation of brucella bacterium shell according to the invention
The brucella bacterium shell of embodiment 1 preparation through physiological saline washing 3 times and resuspended, is observed with transmission electron microscope after the fixed and stained.The result sees Fig. 4 and Fig. 5; Brucella bacterium shell according to the invention keeps the elementary cell form of bacterium; Has complete bacterial outer membrane structure; But, near the bacteriolyze duct, can observe effusive kytoplasm content (Fig. 5 black shade part) owing to the entocyte outflow makes cell surface that obvious shrinkage take place.And the brucella S2 bacterial strain of normal morphology is full, oval, and cell contains the kytoplasm content, sees Fig. 6 and Fig. 7.
Embodiment 6: the safety testing of brucella bacterium shell according to the invention
Select 30 of the Healthy female BALB/c mouse in age in 6-8 week (heavily about 18-20g) for use, be divided into 3 groups at random, 10 every group.The 1st group of abdominal injection brucella S2 bacterial strain attenuated live vaccines, dosage is 10
7CFU/ mouse; The brucella bacterium shell of the 2nd group of abdominal injection embodiment 1 preparation, dosage is 10
8CFU/ mouse (being equivalent to original viable bacteria amount); The 3rd group of isopyknic physiological saline of abdominal injection is as negative control.Observed the mouse body weight change and the mental status in continuous 14 days, and got spleen at last and observe changes in weight.
The result shows that the mouse mean body weight changes as shown in Figure 8.First group of mouse shows One's spirits are drooping, seriously alarms hair, body weight obviously descend the degradation physiological phenomenon, begins to occur dead on the 2nd day from injecting the back, is total to dead 5 to the 6th day.The 2nd group normal with the growth of negative control group mouse, and the body weight change curve is consistent, the no phenomena of mortality.
Inoculate after 14 days, catch and kill all mouse, take by weighing every group of average spleen weight of mouse.The result sees table 1
The mouse spleen weight in average was respectively organized in table 1 inoculation in back 14 days
| The 1st group | The 2nd group | The 3rd group | |
| Average spleen weight (g/ only) | 0.406 | 0.228 | 0.216 |
Can know that by table 1 the 1st group of mouse spleen weight obviously increases, show as extremely enlargement, and the 2nd group with negative control (the 3rd group) to organize mouse similar, spleen does not show considerable change.Synthesizing map 8 can know that with table 1 result the security of brucella bacterium shell according to the invention is apparently higher than existing viable bacteria attenuated vaccine.
Embodiment 7: serum IgG antibody detects after the brucella bacterium shell according to the invention immunity
With 30 of the Healthy female BALB/c mouse in age in 6-8 week (heavily about 18-20g), be divided into 3 groups at random, 10 every group.The 1st group of abdominal injection inoculation brucella S2 bacterial strain attenuated live vaccines (10
6CFU/ mouse), the brucella bacterium shell (10 of the 2nd group of abdominal injection inoculation embodiment 1 preparation
7CFU/ mouse is equivalent to former viable bacteria amount), the 3rd group of abdominal injection inoculated isopyknic physiological saline as negative control group.Whenever at a distance from the docking blood sampling of 1 week, utilize the ELISA method to detect IgG antibody titer in the serum after the immunization.Envelope antigen is brucella vaccine strain S2, and two is anti-for horseradish enzyme labelling goat anti-mouse IgG, develops the color with O-Phenylene Diamine (OPD).Place microplate reader to measure the 495nm absorbance, observe IgG antibody growth and decline rule.The result sees shown in Figure 9; Immunization is after 2 weeks; The 1st group of antibody titers is high than the 2nd group, but the 3rd and 4 all detected results show its no significant difference, illustrates that brucella bacterium shell according to the invention and brucella S2 bacterial strain attenuated live vaccines have the effect that similar immunostimulation produces serum antibody.
Embodiment 8: brucella bacterium shell immanoprotection action test according to the invention
With 60 of the Healthy female BALB/c mouse in age in 6-8 week (heavily about 18-20g), be divided into 3 groups at random, 20 every group.The 1st group of abdominal injection inoculation brucella S2 bacterial strain attenuated live vaccines (10
6CFU/ mouse), the brucella bacterium shell (10 of the 2nd group of abdominal injection inoculation embodiment 1 preparation
7CFU/ mouse is equivalent to former viable bacteria amount), the 3rd group of abdominal injection inoculated isopyknic physiological saline as negative control group.Just exempt from 4 all backs booster immunizations, attack poison with brucella S2 bacterial strain through abdominal injection after 2 weeks again, attacking the toxic agent amount is 10
8CFU/ mouse observed the protection ratio of vaccine to mouse, and the result sees table 2.
Table 2 is respectively organized bacterium liquid and is attacked the protection ratio of poison back to mouse
| Group | Immune mouse | Survival mice | Survival rate |
| The |
20 | 5 | 25％ |
| The |
20 | 13 | 65％ |
| Negative control group (the 3rd group) | 20 | 0 | 0 |
Can know that by table 2 brucella bacterium shell protection effect according to the invention is apparently higher than the protection effect of brucella S2 bacterial strain attenuated live vaccines.
Embodiment 9: prepare brucella bacterium shell according to the invention
Use the forward primer LE-F of nucleotide sequence shown in SEQ ID NO:4 and shown in SEQ ID NO:5 the reverse primer LE-R of nucleotide sequence from phage PhiX174 (available from MBI company), increase and obtain the lysis genes E of nucleotide sequence shown in SEQ ID NO:1;
To shown in SEQ ID NO:1, be cloned among the plasmid pBV220 (giving birth to worker's biotechnology company limited) by the lysis genes E of nucleotide sequence, obtain recombinant plasmid pBV220::E available from Shanghai;
Use the forward primer BRU1 of nucleotide sequence shown in SEQ ID NO:2 and the reverse primer BRU2 amplification recombinant plasmid pBV220::E of nucleotide sequence shown in SEQ ID NO:3; Product after the amplification is connected among the plasmid pBBR1MCS-2 between the SpeI and SalI restriction enzyme site, obtains recombinant plasmid pBBR1MCS-2-E;
Recombinant plasmid pBBR1MCS-2-E is transformed in the brucella M5 bacterial strain and obtains recombinant bacterial strain, recombinant bacterial strain is bred at 28 ℃ be cultured to its OD
600Value is to be warming up to 42 ℃ rapidly after 1.0 to carry out inducing culture to its OD
600Value stabilization, centrifugal then collection thalline, the washing back promptly gets with 5% sodium-chlor freeze thawing.
Method according to embodiment 2-embodiment 8 is carried out the correlation detection analysis to the brucella bacterium shell that present embodiment prepares, and detected result is equal to the correlated results of the brucella bacterium shell of embodiment 1 preparation mutually, all is better than existing brucella bacterial strain attenuated live vaccines.
Embodiment 10: prepare brucella bacterium shell according to the invention
Use the forward primer LE-F of nucleotide sequence shown in SEQ ID NO:4 and shown in SEQ ID NO:5 the reverse primer LE-R of nucleotide sequence from phage PhiX174 (available from MBI company), increase and obtain the lysis genes E of nucleotide sequence shown in SEQ ID NO:1;
To shown in SEQ ID NO:1, be cloned among the plasmid pBV220 (giving birth to worker's biotechnology company limited) by the lysis genes E of nucleotide sequence, obtain recombinant plasmid pBV220::E available from Shanghai;
Use the forward primer BRU1 of nucleotide sequence shown in SEQ ID NO:2 and the reverse primer BRU2 amplification recombinant plasmid pBV220::E of nucleotide sequence shown in SEQ ID NO:3; Product after the amplification is connected among the plasmid pBBR1MCS-2 between the SpeI and SalI restriction enzyme site, obtains recombinant plasmid pBBR1MCS-2-E;
Recombinant plasmid pBBR1MCS-2-E is transformed in the brucella 104M bacterial strain and obtains recombinant bacterial strain, recombinant bacterial strain is bred at 28 ℃ be cultured to its OD
600Value is to be warming up to 42 ℃ rapidly after 1.0 to carry out inducing culture to its OD
600Value stabilization, centrifugal then collection thalline, the washing back promptly gets with 5% sodium-chlor freeze thawing.
Method according to embodiment 2-embodiment 8 is carried out the correlation detection analysis to the brucella bacterium shell that present embodiment prepares, and detected result is equal to the correlated results of the brucella bacterium shell of embodiment 1 preparation mutually, all is better than existing brucella bacterial strain attenuated live vaccines.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be considered as protection scope of the present invention.
Claims (10)
1. the preparation method of a brucella bacterium shell is characterized in that, comprising:
Step 1, will shown in SEQ ID NO:1, be cloned among the plasmid pBV220 by the lysis genes E of nucleotide sequence, obtain recombinant plasmid pBV220::E;
Step 2, use the forward primer BRU1 of nucleotide sequence shown in SEQ ID NO:2 and the reverse primer BRU2 amplification recombinant plasmid pBV220::E of nucleotide sequence shown in SEQ ID NO:3; Product after the amplification is connected among the plasmid pBBR1MCS-2 between the SpeI and SalI restriction enzyme site, obtains recombinant plasmid pBBR1MCS-2-E;
Step 3, recombinant plasmid pBBR1MCS-2-E are transformed in the brucella bacterial strain and obtain recombinant bacterial strain, recombinant bacterial strain is bred at 25-30 ℃ be cultured to its OD
600Value is carried out inducing culture to its OD for being warming up to 37-45 ℃ rapidly behind the 0.8-1.2
600Value stabilization, centrifugal then collection thalline, the washing back promptly gets with the hypertonic solution freeze thawing.
2. according to the said preparation method of claim 1, it is characterized in that the said lysis genes E of step 1 is prepared by following method:
Use the forward primer LE-F of nucleotide sequence shown in SEQ ID NO:4 and shown in SEQ ID NO:5 the reverse primer LE-R of nucleotide sequence from phage PhiX174, increase and obtain lysis genes E.
3. according to the said preparation method of claim 1, it is characterized in that the said electricity that is converted into of step 3 transforms.
4. according to the said preparation method of claim 1, it is characterized in that the said brucella bacterial strain of step 3 is brucella S2 bacterial strain, brucella M5 bacterial strain, brucella RB51 bacterial strain or brucella 104M bacterial strain.
5. according to the said preparation method of claim 1, it is characterized in that the said breeding culture temperature of step 3 is 28 ℃.
6. according to the said preparation method of claim 1, it is characterized in that the said inducing culture temperature of step 3 is 42 ℃.
7. according to the said preparation method of claim 1, it is characterized in that the said hypertonic solution of step 3 is 5% sodium chloride solution.
8. by any brucella bacterium shell that said preparation method prepares of claim 1-7.
9. the application of the said brucella bacterium of claim 8 shell in the vaccine of preparation prevention brucellosis.
10. according to the said application of claim 9, it is characterized in that said vaccine comprises the claim 7 of significant quantity said brucella bacterium shell and pharmaceutically acceptable carrier.
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| CN105255933A (en) * | 2015-10-22 | 2016-01-20 | 中国农业科学院上海兽医研究所 | pTM series plasmid vector and construction method thereof and application in brucella expressed exogenous genes |
| CN105255933B (en) * | 2015-10-22 | 2019-01-11 | 中国农业科学院上海兽医研究所 | PTM series plasmids carrier, its construction method and the application in the bacterium expression alien gene of cloth Shandong |
| CN108690823A (en) * | 2018-04-25 | 2018-10-23 | 内蒙古华希生物科技有限公司 | A kind of brucella ghost combination vaccine loading DNA |
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