CN116622740A - Gene sequence and protein for encoding streptococcus suis protective antigen and application thereof - Google Patents
Gene sequence and protein for encoding streptococcus suis protective antigen and application thereof Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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Abstract
The invention relates to a gene sequence and protein for encoding streptococcus suis protective antigen and application thereof. The invention synthesizes the nucleotide sequence shown in SEQ.No.1 artificially; the primer pair with EcoRI and XhoI cleavage sites is used for amplifying a nucleotide sequence to obtain a target gene PCR product and pET-28a positive plasmid which are connected after double cleavage treatment by EcoRI and XhoI enzyme, and then the expression of recombinant protein aa-ABC is induced to obtain protective antigen aa-ABC, which can obviously improve the antibody level of mice and provide better immune protection effect for type 2, type 7 and type 9 streptococcus suis infection.
Description
Technical Field
The invention relates to a gene sequence and protein for encoding streptococcus suis protective antigen and application thereof.
Background
Streptococcus suis is widely distributed in nature and has various serotypes, so that pigs, horses, cattle, sheep and poultry can become infectious hosts, and human infection can be caused, and the streptococcus suis is an important zoonotic. Streptococcus suis is a gram-positive facultative anaerobe that is a conditional pathogen that is widely found in the respiratory, digestive and reproductive tracts of hosts. In early infection, the host may develop fever, listlessness, etc. The main clinical symptoms are manifested by regional suppurative symptoms of lymph nodes and septicemia. Currently, streptococcus suis is classified into 35 serotypes (types 1/2, 1-34) according to capsular polysaccharides, but a considerable number of streptococcus suis strains cannot be typed, wherein streptococcus suis types 2, 7 and 9 are the serotypes with common pathogenicity, and streptococcus suis type 2 is the most clinically separated serotype and has the highest virulence.
The treatment of streptococcus suis is mainly dependent on antibiotics at present, but the long-term use of antibiotics can lead to the generation of drug resistance of strains, so that more scientific methods should be adopted for treating streptococcus suis, along with the continuous development of biotechnology, the development of genetic engineering vaccines is more and more emphasized, and one important step of the genetic engineering vaccine subunit vaccine of streptococcus suis is the screening of protective antigens.
Liu Lina by comparing and analyzing genes of 4 strains of streptococcus suis in China and the Netherlands, the similarity of the RfeA genes is found to be 100%, and the genes are presumed to be very conservative. After mice are immunized by the prokaryotic expressed protein, the mice are challenged by the type 2 virulent strain OSZYH33, the result shows that the survival rate of the mice in the RfeA immunized group is 90 percent, which is obviously higher than that of the mice in the control group, and the protein has better immunogenicity. After prokaryotic expression, immunogenicity analysis shows that 80% protection can be provided when SS2 is detoxified at high dose, and the homologous HP0245 gene and the highly conserved sequence encoding extracellular skin segment exist in most SS standard strains, so that HP0245 can become candidate protein for developing subunit vaccine. Enolase has been shown to be expressed in all serotypes of Streptococcus suis and is a highly conserved sequence. Studies have demonstrated that Enolase emulsified with a specific adjuvant induces a significant humoral immune response in a mouse model and can provide effective protection against lethal doses of SS2 and SS7 infection.
Disclosure of Invention
In the prior art, the streptococcus suis protective antigen only has a protective effect on type 2 virulent strains and has poor effects on other types of streptococcus suis, and in order to overcome the defects of the technology, the invention provides a streptococcus suis broad-spectrum protective antigen (amino acid ABC transporter substrate-binding protein) aa-ABC and a gene sequence thereof, and discloses a preparation method and application thereof. The invention synthesizes the nucleotide sequence shown in SEQ.No.1 artificially; the nucleotide sequence is amplified by using a primer pair with EcoRI and XhoI cleavage sites, the target gene PCR product and pET-28a positive plasmid are connected after double cleavage treatment by using EcoRI and XhoI enzyme, and then the expression of recombinant protein aa-ABC is induced, so that protective antigen aa-ABC is obtained, and the antibody level of a mouse can be remarkably improved.
The technical effects of the invention are realized by the following technical scheme:
the invention aims to provide a gene sequence for encoding streptococcus suis protective antigen and protein thereof, wherein the gene sequence encodes streptococcus suis broad-spectrum protective antigen (amino acid ABC transporter substrate-binding protein), which is named aa-ABC, and the nucleotide encoding sequence is shown as SEQ.No.1. The nucleotide coding sequence is synthesized by Shanghai Paenox biological Co. The invention also provides a streptococcus suis protective antigen protein corresponding to the gene sequence.
The second object of the present invention is to provide a method for preparing the protective antigen of streptococcus suis, comprising the following steps:
1) Artificially synthesizing a streptococcus suis protective antigen aa-ABC nucleotide sequence shown in SEQ.No.1; amplifying the nucleotide sequence by using a primer pair with EcoRI and XhoI cleavage sites, wherein the forward primer is shown as SEQ.No. 2, and the reverse primer is shown as SEQ.No. 3; the amplified product is subjected to agarose gel electrophoresis with concentration of 1%, and then is recovered and purified by using an agarose gel DNA recovery kit, so that a target gene PCR product is obtained;
2) Constructing a recombinant plasmid pET28a-aa-ABC: performing double digestion treatment on the target gene PCR product and the pET-28a positive plasmid by using EcoRI and XhoI enzymes respectively; recovering aa-ABC gene subjected to double enzyme digestion treatment and pET-28a positive plasmid, and carrying out connection reaction in a metal bath at 16 ℃ overnight;
3) Sequencing and identifying a connection product, then, transforming the connection product into escherichia coli BL21 (DE 3), transferring the transformed competent cells into an LB liquid culture medium with Kan resistance for gradual culture, wherein the culture temperature is 37 ℃;
4) The culture temperature, the culture time and the concentration of IPTG are controlled to induce the expression of recombinant protein, supernatant is collected and then purified and enriched by a nickel column to remove salt, so that aa-ABC antigen of the recombinant protein with the size of 32kDa is obtained, and the antigenicity of the aa-ABC antigen is verified by utilizing Western Blot.
The preparation method of the streptococcus suis protective antigen aa-ABC, wherein the composition of the amplification system in the step 1) is as follows: 25 mu L of LA Taq enzyme, 1 mu L of forward primer and 1 mu L of reverse primer; template 2. Mu.L, ddH 2 O21. Mu.L, PCR amplification conditions were: pre-denaturation at 95℃for 2min;95℃30s,55℃30s,72℃1min,35 cycles, 72℃extension 10min.
The preparation method of the streptococcus suis protective antigen aa-ABC comprises the following steps of: 10 XBuffer 2. Mu.L, aa-ABC Gene 9. Mu.L, ecoRI enzyme 0.5. Mu.L of XhoI enzyme 0.5. Mu.L, ddH 2 O8 μl; the cleavage system for pET-28a plasmid consisted of 10 XBuffer 2. Mu.L, pET-28a plasmid 9. Mu.L, ecoRI enzyme 0.5. Mu.L, xhoI enzyme 0.5. Mu.L, ddH 2 O 8μL。
The preparation method of the streptococcus suis protective antigen aa-ABC comprises the following steps of: 10 XBuffer 1. Mu.L, aa-ABC gene 7. Mu.L, pET-28a plasmid 1.5. Mu.L, T4 DNA ligase 0.5. Mu.L.
The preparation method of the streptococcus suis protective antigen aa-ABC, which is described above, wherein the sequencing identification step in the step 3) specifically comprises the following steps: the ligation reaction product is transformed into competent cells DH5 alpha, the transformed bacterial liquid is coated on an LB agar plate for culture, single bacterial colony is picked up and transferred into an LB liquid culture medium, bacterial liquid is collected, plasmids are extracted for sequencing, and comparison analysis is carried out with target fragments.
In the preparation method of the streptococcus suis protective antigen aa-ABC, the transformation and identification of the connection product specifically comprises the following steps:
adding the connection product into the defreezed competent cells, performing heat shock for 90s in a water bath at 42 ℃, and immediately placing on ice for 2min; adding LB culture medium at room temperature, and shake culturing at 200rpm and 37deg.C for 1 hr; centrifuging the culture medium of competent cells at 8000rpm for 2min, discarding supernatant, re-suspending thallus with culture medium, uniformly spreading 50 μl of thallus on LB agar plate of kanamycin, and culturing at 37deg.C under inversion overnight; picking single colony on the flat plate, transferring to LB liquid culture medium with kanamycin resistance, and shaking overnight at 37 ℃ for culture; and (3) collecting bacterial liquid, extracting plasmids by using a kit after bacterial liquid PCR, sequencing, and comparing and analyzing the obtained sequences with a target fragment by using Snapgene software.
The third object of the present invention is to protect the use of the above-mentioned proteins for providing immune protection against serotype 2, 7, 9 streptococcus suis infections. The embodiment of the invention shows that the recombinant protein can obviously improve the expression level of the antibody Ig G of a host animal. The recombinant protein can obviously improve the expression level of host animal serum and visceral cytokines IL-4 and IFN-gamma.
Compared with the protective antigen in the prior art, the invention has wider immune protection effect on streptococcus suis infection, has obvious cytokine regulation and immune enhancement effects, and has the following technical advantages:
1) The recombinant protein of the invention not only has good protective effect on the serotype 2 streptococcus suis infection, but also has good immune protective effect on the type 7 and type 9 streptococcus suis infection. Example 2 of the present invention shows that the recombinant protein can significantly increase the expression level of the antibody Ig G in a host animal.
2) The recombinant protein can regulate the effect of serum and organ cytokines, and the embodiment 2 of the invention shows that the recombinant protein can obviously improve the expression level of host animal serum and organ cytokines IL-4 and IFN-gamma.
Drawings
Expression and purification of aa-ABC protein, given: m is Protein marker;1, pet28a empty plasmid; 2 protein aa-ABC expression supernatant; 3, expressing inclusion body lysate by protein aa-ABC; 4,5 purified aa-ABC protein.
FIG. 2 aa-ABC monitoring of antibody levels after immunization
FIG. 3. Detection of IL-4, IFN-gamma secretion levels in mice lung, spleen and serum.
FIG. 4 toxicity counteracting protection survival curves for Streptococcus suis types 2, 7 and 9
Detailed Description
The invention is further described below by means of specific examples, which do not in any way limit the scope of the patent protection of the invention. The protection scope of the invention is defined by the claims.
The methods for extracting the type 2, type 7 and type 9 serotype streptococcus suis DNA used in the examples of the present invention are as follows:
(1) Scribing and resuscitating the streptococcus suis type 2, 7 and 9 preserved at-80 ℃ on a 5% Columbia blood agar culture medium, culturing overnight in a 37 ℃ incubator, picking up a monoclonal and inoculating to a TSB culture medium, and shake culturing at 200rpm at 37 ℃ for 12 hours;
(2) Centrifuging 200 μL of bacterial liquid at 8000rpm for 5min, and discarding supernatant;
(3) Re-suspending the cells with 500. Mu.L of sterile TE lysate, centrifuging at 8000rpm for 5min, and discarding the supernatant;
(4) The cells were resuspended in 100. Mu.L of sterile TE lysate, boiled in a boiling water bath for 15min, ice-bath for 5min, centrifuged at 12000rpm for 10min, and the supernatant was aspirated as a template.
The method for extracting the plasmid in the embodiment of the invention is as follows: and resuscitating DH5 alpha frozen bacteria containing the expression vector pET-28a, and picking a monoclonal colony for expansion culture. Empty plasmids were extracted according to the AXYGEN plasmid miniprep kit instructions.
(1) Taking 5mL of the overnight cultured bacterial liquid, centrifuging 12000 Xg for 1min, and discarding the supernatant;
(2) Adding 250 mu L of S1 Buffer (RNase A is added) into a centrifuge tube with the bacterial pellet, suspending and precipitating, and leaving no bacterial block;
(3) Adding 250 mu L of S2 Buffer into a centrifuge tube, gently and fully turning over for 4-6 times up and down, and uniformly mixing to ensure that thalli are fully cracked until a transparent solution is formed, wherein the step is not suitable for more than 5min;
(4) Adding 350 mu L of S3 Buffer into the centrifuge tube, gently and fully overturning up and down for 6-8 times, and centrifuging for 1min at 12000 Xg;
(5) The supernatant from step 4 was aspirated and transferred to a preparation tube (as in 2mL, provided in the kit) and centrifuged at 12000 Xg for 1min to discard the filtrate;
(6) Placing the preparation tube into a centrifuge tube, adding 500 mu L of W1 Buffer, centrifuging 12000 Xg for 1min, and discarding the filtrate;
(7) Placing the preparation tube into a centrifuge tube, adding 700 mu L of W2 Buffer (absolute ethyl alcohol is added), centrifuging 12000 Xg for 1min, discarding the filtrate, and repeating the step once;
(8) The preparation tube was put back into a 2mL centrifuge tube, and 12000 Xg was centrifuged for 1min, and the rinsing liquid remaining in the column was removed;
(9) Placing the preparation tube into a new 1.5mL centrifuge tube, adding 60-80 mu L of Eluent (heated to 65 ℃ in advance) in the center of the preparation tube film, standing for 1min at room temperature, and centrifuging for 1min at 12000 Xg;
(10) The plasmid concentration was measured and stored at-20 ℃.
Example 1 Streptococcus suis protective antigen aa-ABC Gene sequence and preparation method thereof
The expression method of the streptococcus suis protective antigen aa-ABC gene sequence specifically comprises the following steps: a. primer design and amplification of target gene
Artificially synthesizing a nucleotide sequence shown in SEQ.No.1 as a template, and amplifying the nucleotide sequence by using a primer pair with EcoRI and XhoI cleavage sites, wherein a forward primer is shown in SEQ.No.2, and a reverse primer is shown in SEQ.No.3; the aa-ABC gene primer was synthesized by Shanghai Paeno Bio Inc.
The streptococcus suis type 2 is used as a template for amplifying the gene aa-ABC, and the amplification system is as follows (50 mu L):
the PCR amplification conditions were: pre-denaturation at 95℃for 2min;95℃30s,55℃30s,72℃1min,35 cycles, 72℃extension 10min. After 1% agarose gel electrophoresis of amplified products, the target gene is recovered and purified by using an agarose gel DNA recovery kit, and the recovered target gene is stored at-20 ℃.
b. Ligation, construction of pET28a-aa-ABC recombinant plasmid
First, the PCR product of the target gene and pET-28a positive plasmid were subjected to double digestion with EcoRI and XhoI enzymes, wherein the PCR product digestion system (20. Mu.L) of the target gene was
The pET-28a positive plasmid enzyme digestion system (20 mu L) is
The prepared enzyme digestion system is placed in a water bath kettle at 37 ℃ for reaction for 2 hours, then 1% agarose gel electrophoresis is carried out, the OMEGA agarose gel DNA recovery kit is utilized for recovering and purifying the target gene, and the target gene is stored at-20 ℃ for standby after the concentration is measured.
The recovered double digested aa-ABC gene and pET-28a positive plasmid were ligated overnight in a metal bath at 16 ℃. The ligation system (10. Mu.L) was:
c. recombinant plasmid transformation and identification
Transforming the recombinant plasmid into competent cells DH5 alpha and identifying, wherein the specific steps are as follows:
(1) Removing competent cells DH5 alpha from the refrigerator at-80 ℃ and thawing on ice;
(2) Adding 10 μl of the ligation product into 100 μl of competent cells, mixing with the wall of a gentle bomb, mixing without shaking, and incubating on ice for 30min;
(3) Heat shock in a water bath at a temperature of 42 ℃ for 90s, and immediately placing on ice for 2min;
(4) 600. Mu.L of LB medium balanced to room temperature is added, and shaking culture is carried out for 1h at 200rpm and 37 ℃;
(5) Centrifuging the cultured competent cell culture solution at 8000rpm for 2min, discarding supernatant, resuspending thallus with 100-120 μl of the rest culture medium, uniformly coating 50 μl of thallus on LB agar plate of kanamycin, and inverting at 37deg.C
Culturing overnight in an incubator;
(6) Single colony on LB agar plate is selected and transferred to 3mL LB liquid medium (containing 50 mug/mL kanamycin) for overnight culture at 37 ℃ with shaking;
(7) Bacterial liquid is collected, plasmid extraction kit of OMEGA company is used for extracting plasmid by bacterial liquid PCR, and specific operation steps refer to the operation instructions of the plasmid kit. The extracted plasmid is sent to the Hepersonoo biosis Co., ltd for sequencing, and the obtained sequence is compared and analyzed with the target fragment by utilizing Snapgene software.
d. Prokaryotic expression and detection of target gene
(1) Transformation of a successfully constructed pET28a-aa-ABC recombinant plasmid into competent cell BL21 (DE 3), specific procedures reference competent cell BL21 (DE 3) instructions, selection of single colonies for transfer to 5mL Kan resistance (final concentration 50)
Mu g/mL) of the culture medium was cultured at 37℃for 12 hours at 200 rpm;
(2) The prepared bacterial liquid was transferred to 10mL of LB liquid medium with Kan resistance (final concentration: 50. Mu.g/mL) at a ratio of 1:100, and cultured at 37℃and 200rpm to OD 600nm Respectively adding IPTG with proper final concentration when the value is between 0.6 and 0.8, optimizing conditions such as induced temperature, time, rotating speed and the like, and expressing protein in supernatant;
(3) The induced and uninduced BL21 (DE 3) and positive recombinant containing only pET-28a empty vector and the supernatant and precipitate of the positive recombinant after induction were subjected to SDS-PAGE analysis, respectively.
(4) Washing the electrophoresis rubber plate with neutral detergent, rinsing with double distilled water, and air drying to obtain 12% separating rubber;
(5) And adding 15 mu L of the prepared sample into a sample loading hole, stabilizing the voltage for 80V electrophoresis, and increasing the voltage to 120V after the front edge of the bromophenol blue serving as an indicator enters the separation gel to continue electrophoresis until the indicator migrates to the lower edge of the separation gel.
(6) After electrophoresis, the gel was removed, placed in a large petri dish with a lid, poured into coomassie brilliant blue staining solution until the gel was immersed, and stained on a horizontal shaker for 30min. The staining solution was recovered, the gel was rinsed with a small amount of water and decolorized on a horizontal shaker until the blue background disappeared.
e. Protein purification
By conditional fumbling, the expression form of the protein aa-ABC was found to be a soluble protein, and the purification steps were as follows:
(1) The overnight cultured bacterial liquid (containing pET28a-aa-ABC recombinant plasmid) was transferred to 200mL of LB liquid medium containing Kan (final concentration 50. Mu.g/mL) at a ratio of 1:100, and cultured at 37℃and 200rpm to OD 600nm A value of 0.6 to 0.8A compartment;
(2) Through condition searching, the optimal expression condition of the protein aa-ABC is 37 ℃ and is induced at 200rpm overnight, and the final concentration is 1
mmol/mL IPTG;
(3) Centrifuging the induced bacterial liquid at 8000rpm for 5min to collect bacteria, blowing and mixing the bacterial mass of protein aa-ABC with 40mL of Lysis Buffer, performing ultrasonic crushing treatment until the solution becomes clear, centrifuging at 8000rpm for 30min to separate supernatant and precipitate, and collecting supernatant;
(4) Adding 8mL of Lysis Buffer for balancing chromatography medium, loading a protein clarified sample containing a polyhistidine tag into a column, controlling the flow rate to be 0.5-1 mL/min, and collecting effluent for subsequent analysis;
(5) Washing protein aa-ABC with 10mL of 250mM solution Buffer, and performing gradient Elution at a flow rate of 0.5-1
mL/min。
f. Protein enrichment and ultrafiltration desalination
(1) Filling UP water into a 10kDa ultrafiltration tube and a 3kDa ultrafiltration tube, and precooling on ice for 10min;
(2) Adding purified protein aa-ABC into a 10kDa ultrafiltration tube, centrifuging at 6000rpm for 15min, and continuously adding 1 XPBS during ultrafiltration to dilute and desalt;
(3) Protein SDS-PAGE electrophoresis analysis after desalting and ultrafiltration is carried out, protein concentration is measured, split charging is carried out, and the split charging and freezing are carried out at-80 ℃. The result of the electrophoresis analysis is shown in FIG. 1.
g. Recombinant protein antigenicity analysis
(1) Performing SDS-PAGE on each purified recombinant protein, and then transferring the purified recombinant protein onto an NC film in a constant-current 240mA and 2h ice bath;
(2) Sealing with 5% skimmed milk for 2 hr, and washing with PBST 3 times for 10min each time;
(3) Streptococcus suis type 2 positive serum (1:200) was used as primary antibody, incubated overnight at 4℃and then washed 3 times with 10 times with PBST
min；
(4) HRP-labeled goat anti-rabbit IgG as secondary antibody (1:10000), incubated at room temperature for 50min, then washed 3 times with PBST for 10min each;
(5) And (3) developing by an ECL method.
Example 2 mouse infection experiment
a. Rejuvenation of strains
(1) Resuscitates SS2, SS7 and SS9 strains frozen at-80 ℃ on 5% of blood plates respectively by streaking, and cultures overnight in a constant temperature incubator at 37 ℃;
(2) The monoclonal on the blood plate is picked up and cultured overnight at 37 ℃ and 200rpm in 5mL TSB culture medium;
(3) Overnight cultured SS2, SS7 and SS9 strain 1:100 inoculated in 10mL of TSB culture medium at 37 ℃ and 200 DEG C
Culturing at rpm for 6h, centrifuging at 8000rpm for 5min to collect thallus, re-suspending with PBS, washing for 3 times, and adjusting bacterial concentration with PBS to 1×109CFU/mL, 2×109CFU/mL, and 1.8X109 CFU/mL, respectively, and intraperitoneal injection of 200 per mouse
μL；
(4) Separating bacteria from dying mice lung for amplifying bacterial condition in 12-24 h, performing PCR identification, wherein the forward identification primer of SS2 serotype is shown as SEQ. NO.4, and the reverse identification primer of SS2 serotype is shown as follows
SEQ.NO. 5; the forward identification primer of the serotype of the SS7 is shown as SEQ.NO.6, and the reverse identification primer of the serotype of the SS7 is shown as SEQ.NO. 7; the forward identification primer of the serotype of the SS9 is shown as SEQ. NO.8, and the reverse identification primer of the serotype of the SS9 is shown as SEQ. NO. 9.
(5) The PCR reaction system was 20. Mu.L, 2 XTaq Mix 10. Mu.L, each 1. Mu.L of the upstream primer, each 1. Mu.L of the downstream primer, each 2. Mu.L of the template, and the remaining insufficient volume was complemented with ddH 2O. The PCR conditions were as follows: pre-denaturation at 95 ℃ for 5min;30 cycles (denaturation at 94℃for 30s, annealing at 56℃for 50s, extension at 72℃for 50 s); extending at 72℃for 7min. The products after PCR were electrophoresed for 30min on a 1% agarose gel at 120V voltage.
b. Determination of half-number lethal dose (LD 50) in mice
(1) SS2, SS7, SS9 strains after rejuvenation in mice were grown at 1:100 inoculated in TSB, cultured at 37℃and 200rpm for 6
h，OD 600 0.6 to 0.8;
(2) Centrifuging at 8000rpm for 5min to collect thallus, and washing with PBS3 times later, the bacteria concentration is adjusted to be 2.0X10 respectively 9
CFU/mL、1.6×10 9 CFU/mL、2.0×10 9 CFU/mL, diluting bacterial suspension 10 times with PBS as diluent, taking 10 0 、10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Group abdominal cavity inoculation is carried out on 200 mu L/mouse, 8 mice in each group are injected with PBS with equal quantity, each experimental group is fed with PBS with equal quantity, the morbidity and mortality of the mice are observed at regular time, and the mice are observed continuously for 7 days;
(1) Calculation of half-lethal dose LD according to Reed-Muench method 50 Calculate the half-Lethal Dose (LD) 50 ) The formula of (2) is as follows:
distance ratio r= (greater than 50% mortality a-50)/(greater than 50% mortality a-less than 50% mortality B)
lgLD 50 Log + distance ratio x dilution times =inverse mortality dilution above 50%
According to the above formula, 50% of mice die when the stock solution is diluted to a certain multiple, and LD of SS2 can be obtained according to experimental data 50 The method comprises the following steps: 3.56X10 7 CFU/mL; LD of SS7 50 4.02X10 7 CFU/mL; LD of SS9 50 Is 7.88×10 7 CFU/mL。
c. Immunization of mice
66 3-week-old BALB/c were randomly divided into 6 groups of 11 mice, 3 of which were immunized with aa-ABC at 100. Mu.L (1 mg/mL), and the remaining 3 groups were inoculated with 100. Mu.L of PBS as a negative control group. The individual proteins were mixed and emulsified 1:1 with Freund's incomplete adjuvant prior to immunization and injected subcutaneously at multiple points. The first immunization was followed by a second immunization at a dose of 100 μg after 14 days.
d. Toxicity attack experiment of streptococcus suis 2, 7 and 9
At 10 days post last immunization, mice immunized with 8-fold LD were used in each group 50 SS2, SS7 and SS9 were challenged by intraperitoneal injection, continuously observed for 7 days, and the onset and death recorded, and fig. 4 shows the toxicity attack protection survival curves of streptococcus suis type 2, type 7 and type 9, and experimental results show that recombinant protein protection was not performedAntigen-immunized mice infected with streptococcus suis die within 36 hours or 48 hours after infection, and the recombinant protein protective antigen can greatly improve the survival rate of the mice infected with streptococcus suis. Among them, the best protective effect is achieved against type 9 Streptococcus suis.
e. Detection of antibody levels in mice
ELISA plates are coated with purified recombinant fusion proteins respectively, negative control is set, the change of antibody titer in serum of mice after immunization is monitored, and the P/N value is more than or equal to 2.1 and is used as a critical value for judging the specificity.
Collecting appropriate amount of whole blood through retroorbital venous plexus, collecting serum of mice at day 13 of primary immunization and day 9 of secondary immunization, standing at room temperature for 2 hr, standing overnight at 4deg.C, centrifuging at 3000rpm at 4deg.C for 15min, collecting serum, and aseptically packaging and storing at-80deg.C for use. The results of the in vivo antibody level determination of the mice on day 13 of the first immunization and on day 9 after the second immunization are shown in FIG. 2. Experimental results show that the IgG level of the mouse antibody immunized by using the protective antigen is equivalent to that of the mouse antibody immunized by not using the protective antigen when the mouse antibody is immunized for the first time, and no obvious difference exists. The IgG level of the mice immunized with the protective antigen is significantly higher than that of the mice immunized without the protective antigen on the 13 th day of the first immunization and the 9 th day of the second immunization, which is higher than that of the mice immunized with the protective antigen on the 13 th day of the first immunization, indicating that the protective antigen of the invention can significantly improve the antibody expression level of the mice.
(1) Taking a 96-hole detachable ELISA plate, respectively coating 100 mu L of protein aa-ABC (final dosage of protein is 1 mu g) diluted by coating liquid on each hole, and coating at 4 ℃ overnight;
(2) Plates were washed 3 times with 0.5% PBS-T wash, drained, 200. Mu.L 1% BSA blocking solution was added to each well and blocked at 37℃for 2h.
(3) Plates were washed 3 times with 0.5% PBS-T wash to drain water;
(4) The ratio is 1:200,1:400,1:800,1:1600,1:3200,1:6400,1:12800,1:25600,
1:51200 and 1:102400 diluted post-immunization serum;
(5) Adding the diluted serum into ELISA plates coated with antigens respectively, wherein each hole is 100 mu L, and 3 compound holes are formed;
(6) After incubation for 1h at 37 ℃, the plates were washed 3 times with 0.5% PBS-T wash, and drained;
(7) 100 mu L of HRP-sheep anti-mouse IgG diluted with 1:10000 is added into each hole, and incubated for 30min at 37 ℃;
(8) Plates were washed 3 times with 0.5% PBS-T wash to drain water;
(9) Adding 100 mu L of TMB color development liquid into each hole, and incubating for 15min at room temperature in a dark place until the reaction is complete;
(10) 50. Mu.L of stop solution was added to each well, and the OD was measured at 450nm in an ELISA reader.
f. Determination of mouse serum and organ cytokines
(1) 3 mice were randomly taken from the immunized and control groups for sterile dissection before post-secondary challenge, and the lungs of the mice were removed and placed in 1mL PBS in advance according to 1:10 (mg: mL) was weighed and recorded (the weight of the EP tube containing 1mL of PBS was previously weighed) and the lung sample were homogenized, and centrifuged at 3000rpm and 4℃for 50℃after homogenization
min, sucking supernatant as much as possible, packaging, and standing at-20deg.C;
(2) IL-4 and IFN-gamma ELISA detection kits were used to detect the levels of IL-4 and IFN-gamma in serum, lung and spleen,
the operation steps are shown in the instruction book in the kit.
The measurement results are shown in FIG. 3. The experimental results show that the expression levels of IL-4 and IFN-gamma were significantly higher in mice immunized with protective antigen than in the control group, both in serum and in the lungs and spleen. This suggests that the protective antigen has the effect of significantly elevating the expression levels of IL-4 and IFN-gamma.
Claims (10)
1.A gene sequence for coding the streptococcus suis protective antigen has a nucleotide sequence shown in SEQ.No.1.
2.A streptococcus suis protective antigen protein corresponding to the gene sequence of claim 1.
3. The method for preparing the streptococcus suis protective antigen as claimed in claim 1, comprising the steps of:
1) Artificially synthesizing a streptococcus suis protective antigen aa-ABC nucleotide sequence shown in SEQ.No.1; the method comprises the steps of carrying out a first treatment on the surface of the Amplifying the nucleotide sequence by using a primer pair with EcoRI and XhoI cleavage sites, wherein the forward primer is shown as SEQ.No. 2, and the reverse primer is shown as SEQ.No. 3; the amplified product is subjected to agarose gel electrophoresis with concentration of 1%, and then is recovered and purified by using an agarose gel DNA recovery kit, so that a target gene PCR product is obtained;
2) Constructing a recombinant plasmid pET28a-aa-ABC: double digestion treatment is carried out on the target gene PCR product and pET-28a positive plasmid by using EcoRI and XhoI enzymes; recovering aa-ABC gene subjected to double enzyme digestion treatment and pET-28a positive plasmid, and carrying out connection reaction in a metal bath at 16 ℃ overnight;
3) Sequencing and identifying a connection product, then, transforming the connection product into escherichia coli BL21, transferring the transformed competent cells into an LB liquid culture medium with Kan resistance for gradual culture, wherein the culture temperature is 37 ℃;
4) The culture temperature, the culture time and the concentration of IPTG are controlled to induce the expression of recombinant protein, the supernatant is collected, and the aa-ABC antigen with the size of 32kDa of the recombinant protein is obtained after nickel column purification, enrichment and desalination, and the antigenicity of the aa-ABC antigen is verified by Western Blot.
4. The method for preparing streptococcus suis protective antigen aa-ABC according to claim 2, wherein the composition of the amplification system in step 1) is as follows: 25 mu L of LA Taq enzyme, 1 mu L of forward primer and 1 mu L of reverse primer; template 2. Mu.L, ddH 2 O21. Mu.L, PCR amplification conditions were: pre-denaturation at 95℃for 2min;95℃30s,55℃30s,72℃1min,35 cycles, 72℃extension 10min.
5. The method for preparing streptococcus suis protective antigen aa-ABC according to claim 2, wherein the composition of the cleavage system of the PCR product of the target gene in step 2) is:10 XBuffer 2. Mu.L, aa-ABC Gene 9. Mu.L, ecoRI enzyme 0.5. Mu.L, xhoI enzyme 0.5. Mu.L, ddH 2 O8 μl; the cleavage system for pET-28a plasmid consisted of 10 XBuffer 2. Mu.L, pET-28a plasmid 9. Mu.L, ecoRI enzyme 0.5. Mu.L, xhoI enzyme 0.5. Mu.L, ddH 2 O 8μL。
6. The method for preparing streptococcus suis protective antigen aa-ABC according to claim 2, wherein the composition of the ligation reaction system in step 2) is: 10 XBuffer 1. Mu.L, aa-ABC gene, 7. Mu.L, pET-28a plasmid 1.5. Mu.L, T4 DNA ligase 0.5. Mu.L.
7. The method for preparing streptococcus suis protective antigen aa-ABC according to claim 2, wherein the step 3) of sequencing and identifying comprises: the ligation reaction product is transformed into competent cells DH5 alpha, the transformed bacterial liquid is coated on an LB agar plate for culture, single bacterial colony is picked up and transferred into an LB liquid culture medium, bacterial liquid is collected, plasmids are extracted for sequencing, and comparison analysis is carried out with target fragments.
8. Use of a streptococcus suis protective antigen protein as claimed in claim 2 to provide immunoprotection against serotype 2, 7, 9 streptococcus suis infections.
9. The use according to claim 8, wherein the streptococcus suis protective antigen protein is capable of significantly increasing the expression level of the antibody Ig G in the host animal.
10. The use according to claim 8, wherein said streptococcus suis protective antigen protein is capable of significantly increasing the expression levels of host animal serum and visceral cytokines IL-4 and IFN- γ.
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