CN101940786B - Application of immunogenic protein gene PilA of haemophilus parasuis - Google Patents
Application of immunogenic protein gene PilA of haemophilus parasuis Download PDFInfo
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- CN101940786B CN101940786B CN200910273436A CN200910273436A CN101940786B CN 101940786 B CN101940786 B CN 101940786B CN 200910273436 A CN200910273436 A CN 200910273436A CN 200910273436 A CN200910273436 A CN 200910273436A CN 101940786 B CN101940786 B CN 101940786B
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Abstract
The invention belongs to the technical field of veterinary microbiology and animal infectious diseases and particularly relates to separation and clone of an immunogenic protein gene of haemophilus parasuis and application of protein coded by the immunogenic protein gene in bacterin and diagnosis. A new immunogenic protein gene pilA is separated from a virulent strain SH0165 of the haemophilus parasuis; the nucleotide sequence of the protein gene is disclosed in the sequence table SEQ-ID-NO:1; 150 amino acids are coded; and the coding product of the gene is the new immunogenic protein gene pilA which can provide effective protective immunity for piglets infected by haemophilus parasuis 0165. The invention also relates to preparation of a recombinant strain of colon bacillus DH5a/Hps-pilA expressing the immunogenic protein gene pilA. The recombinant strain of colon bacillus is conserved in China Center for Type Culture Collection (CCTCC) with conservation number is CCTCC-NO:M 209303.
Description
Technical field
The invention belongs to veterinary microbiology and zoonosis technical field.Be specifically related to separation, clone and its an encoded protein application in vaccine and diagnosis of haemophilus parasuis with immunogenic protein gene.
Background technology
Haemophilus parasuis (Haemophilus parasuis; HPS) being a kind of Gram-negative dialister bacterium of Pasteurellaceae, is the pathogen of haemophilus parasuis sick (
disease).As one of the most serious bacterial infectious disease of current harm world pig industry; The haemophilus parasuis disease can influence the pig at 2 age to 4 monthly ages in week; Piglet is mainly in wean front and back and the morbidity of child care stage; Usually be shown in the pig in 5～8 ages in week, sickness rate is generally 10%～15%, and mortality rate is up to 50% when serious.Main clinic symptoms shows as cough, dyspnea, the row and dead of becoming thin, cross mountains; The major lesions of cuing open inspection shows as polyserositis (fibrinous pleurisy, pericarditis, peritonitis), arthritis and meningitis etc.Haemophilus parasuis also can cause septicemia, and after actute infection, possibly stay sequela, i.e. sow miscarriage, the chronic limping of boar have increased the mortality of boar.In addition; Because the bacterial bearing rate of haemophilus parasuis is very high in the swinery, when viral infectious such as swinery generation Porcine reproductive and respiratory syndrome, pig circular ring virus 2 viral disease, the sick sickness rate of haemophilus parasuis can sharply raise; Endanger even more seriously, cause large quantities of pigs dead.
In recent years, the haemophilus parasuis disease is at the M & M of the high healthy swinery of large-scale pig farm all significantly raise (Cai X, Chen H; Blackall P J, Yin, Z; Wang, L, Liu Z and Jin M.Serological characterization of Haemophilus parasuis isolatesfrom China.Vet Microbiol; 2005,111:231-236).Because this disease breaks out suddenly as a rule, the therapeutical effect of antimicrobial agents such as antibiotic is very limited, and chemical sproof generation is rapid, and therefore, medicine and immunoprophylaxis are the main paties that prevention and control should disease.At present, conventional inactivated vaccine is unique in the world one type of vaccine that can be used for this disease immune protection, has realized commercialization in a plurality of countries that comprise China.These inactivated vaccines generally are prepared from (Baumann G the local popular bacterial strain of 2 or 3 serotypes; Bilkei G.Effect of vaccinatingsows and their piglets on the development of
disease induced by a virulent strain of Haemophilusparasuis serovar 5.Vet Rec; 2002,151:18-21; Bak H, Riising H J.Protection of vaccinated pigs againstexperimental infection with homologous and heterologous Haemophilus parasuis.Vet Rec, 2002; 151:502-505); But the haemophilus parasuis of having identified at present has 15 kinds of serotypes at least, and 12% the separated strain of also having an appointment still can not typing; And country variant and regional popular advantage serotype are not quite similar; Lack between the different serotypes bacterial strain or limited cross immunity protection only can be provided, the cross immunity protective rate of conventional multivalent inactivated vaccine is limited, and immunoprotection (Oliveira S preferably only just can be provided in the geographic swinery that popular bacterial strain serotype conforms to; Pijoan C.Haemophilus parasuis:new trends on diagnosis; Epidemiology and control.Vet Microbiol, 2004b, 99:1-12).Therefore, the evaluation of the conservative protective antigen of haemophilus parasuis is the key problem in technology of this disease new generation vaccine development.Up to the present, also less about the report of haemophilus parasuis protective antigen in the world, one piece of paper that Zhou Mingguang etc. were published on the Vaccine in 2009 has been done four albumen outer membrane protein PalA first; Omp2, the immunogenicity of D15 and hypothetical protein HPS 06257 has been preliminary research (Zhou M, Guo Y; Zhao J, Hu Q, Hu Y; Zhang A, Chen H, Jin M.Identification and characterization of novel immunogenicouter membrane proteins of Haemophilus parasuis serovar 5.Vaccine; 2009,27 (38): 5271-7).
PilA albumen belongs to protein two type excretory system IV type pili (Type IV Pili); PilA albumen is relevant with the picked-up of DNA; The mediation antibacterial is adsorbed in the first step that the mucosal epithelium cell is accomplished invasion in the pathogenic course, the generation of its pili of pilA gene pairs and biofilm and this pathogen played an important role in the epithelial field planting of mammal upper respiratory tract in hemophilus influenza.The PilA recombinant protein vaccine of (2007) Jiang Yayuan such as Cheng Anchun and chicken source Escherichia coli carries out immunity duckling and chickling respectively, can obtain very high ELIAS antibody titer; Through the counteracting toxic substances laboratory observation, this recombiant protein has the host protects effect (Cheng Anchun, Yu Xiaona effectively; Wang Mingshu, Zhu Dekang, Li Ling; Sun Lei; Chen Xiaoyue. duck source Escherichia coli I type pili pilA Prokaryotic Expression and recombiant protein are to the immanoprotection action of strong virus attack. biological engineering journal, 2007,23:440-445; In coral, Zhang Qian, water brook, Yu Zhouliang, Zhao Baohua. the clone of pathogenic chicken colibacillosis pilA gene and outer membrane protein C gene. the biological engineering journal, 2008,24:1561-1567).
Summary of the invention
The purpose of this research is to clone a GFP with immunogenic haemophilus parasuis, utilizes its encoded protein to be applied to develop haemophilus parasuis infected to have in the subunit vaccine and diagnostic reagent of protection.
The present invention is achieved in that
The applicant is cloned into IV type pilin gene pilA from the Local Isolates SH0165 bacterial strain (bacterium source is referring to embodiment 1) of haemophilus parasuis.Find that through order-checking its coding region of GFP pilA of the present invention by 453 base compositions, has the nucleotide sequence shown in the sequence table SEQ ID NO:1.The albumen pilA of the pilA gene code that the process sequence analysis is found finds that through sequence analysis the albumen PilA of pil4 gene code of the present invention is made up of 286 amino acid residues by 150; Have the aminoacid sequence shown in the sequence table SEQ ID NO:1, remove the signal peptide part big or small 15kDa of being of expressing protein estimated molecular weight afterwards.Confirm the PilA albumen that gene pilA of the present invention expresses through prokaryotic expression and indoor bioassay in microorganism, can protective effect be arranged to the bloodthirsty infection of secondary pig, this is just indicating that this albumen can be used to develop the subunit vaccine of haemophilus parasuis.
Said gene sequence provided by the invention is the immunogenic GFP of having of haemophilus parasuis, and this albumen can also be applied in the development for the diagnosis of haemophilus parasuis and vaccine.
The description of more detailed technical scheme reference implementation example part.
Description of drawings
Sequence table SEQ ID NO:1 is the nucleotide sequence and the encoded protein matter sequence thereof of the haemophilus parasuis immunogenic protein gene of separating clone of the present invention;
Fig. 1: the design of graphics and the physical map that are the prokaryotic expression carrier pET28a/Hps-pilA at haemophilus parasuis immunogenic protein gene pilA place in the embodiment of the invention;
Fig. 2: the SDS-PAGE electrophoretic analysis that is haemophilus parasuis immunogenic protein gene pilA expression and purification in reorganization bacterium BL21 in the embodiment of the invention; Among the figure: M: protein molecular weight standard;
Fig. 3: the antibody horizontal behind the assessment immunogenic protein immunity piglet;
Fig. 4: albumen is to the deadly protective rate of piglet immunological;
Specific embodiments
Below narration is embodiment according to embodiments of the present invention.Should be noted that embodiments of the invention have only illustration for the present invention, and do not have restriction.The standard operating instructions of relative dna and employed medicine are all with reference to " molecular cloning experiment guide " described content (referring to Sa nurse Brooker and Russell, 2001, molecular cloning experiment guide, the third edition, Jin Dongyan etc. (translating), Science Press, Beijing).Related other various experimental implementation among the present invention; Be the ordinary skill in the art; Do not have the part that specifies in the literary composition, those of ordinary skill in the art can implement with reference to the various common tool books before the applying date of the present invention, scientific and technical literature or relevant description, handbook etc.
The clone of embodiment 1 haemophilus parasuis SH0165 bacterial strain immunogenic protein gene pilA
(document is seen in the source of this bacterial strain: Yue M, Yang F, Yang J as the source bacterial strain of immunogenic protein gene pilA with haemophilus parasuis SH0165 in the present invention; Bei W, Cai X, Chen L; Dong J, Zhou R, Jin M; Jin Q, Chen H.Complete genome sequence ofHaemophilus parasuis SH0165.J Bacteriol.2009,191 (4): 1359-60)
This bacterial strain is a Local Isolates, serotype 5 types, infected doses this bacterial strain piglet or young pig can fall ill in 4 days or dead, be supper toxic strain
1. the clone of immunogenic protein gene pilA among the haemophilus parasuis SH0165
Utilize the genome of haemophilus parasuis SH0165 to be template; The applicant introduce according to the distinguished sequence of this gene and at two ends restriction enzyme site BamHI and XhoI design forward primer P1 (primer sequence is: 5 '-ATAGGATCCTCTTACACCAGTTACAC-3 ') and downstream primer P2 (primer sequence is: 5 '-ATACTCGAGTTATTGCCTACAGAAATTC-3 '); Carry out pcr amplification to obtain target gene, the system and the program of PCR reaction are following:
25 μ L reaction systems comprise: 2.5 μ L10 * PCR reaction buffer; 1 μ LdNTP (each 2.5mM); 0.5 μ L specificity forward primer P1 (20mM), 0.5 μ L specificity downstream primer P2 (20mM), 1 μ L template (haemophilus parasuis SH0165); 0.25 μ LEx Taq enzyme adds sterilization deionized water to 25 μ L.PCR response parameter and program are: 94 ℃, and 5min, 1 circulation; 94 ℃, 30s, 60 ℃, 30s, 72 ℃, 1min, 30 circulations; 72 ℃, 10min, 1 circulation.
Be connected on the T carrier pMD18-T Simple Vector (available from TaKaRa company) through the T/A clone after the target fragment that increases reclaimed test kit [available from Sangon Biotech (Shanghai) Co., Ltd.] purifying and recovering through the PCR product, target fragment is connected to the recombiant plasmid pET28a/Hps-pilA (see figure 1) that pET28a [available from Merck & Co., Inc. (Shanghai)] obtains containing the pilA full length sequence.Afterwards with this recombinant plasmid transformed escherichia coli (E.coli) DH5 α, and the dull and stereotyped (Kan of coating kalamycin resistance
R, final concentration is 50 μ g/mL).Place 37 ℃ of incubators to cultivate 12-16h resistant panel, son to be transformed is long after a certain size, the screening transformant.With the liquid LB culture medium activation culture of the transformant that screens with 5mL, the extracting plasmid carries out the enzyme action checking then, and the endonuclease bamhi size is consistent with the expection size.With the liquid LB culture medium activation culture of the positive transformant that screens, get the overnight culture sample presentation order-checking (three rich Radix Polygalae biotechnology Co., Ltds accomplish by Beijing) of 1mL at last with 5mL.Sequencing result shows that this gene has the nucleotide sequence shown in sequence table SEQ ID NO:1, and the applicant is pilA (gene of being named in the present invention, is represented with the italic small letter) with this unnamed gene.Through one of this section of software analysis prediction coded sequence codified polypeptide fragment of forming by 116 aminoacid shown in sequence table SEQ ID NO:1; Predict that its molecular weight is 15kDa; The applicant is with its called after PilA (albumen of being named in the present invention, is with roman capitalization expression).
The applicant is with above-mentioned recombination bacillus coli called after escherichia coli (Escherichia coli) DH5 α/Hps-pilA that has transformed recombiant plasmid pET28a/Hps-pilA; In December in 2009 16 days this recombination bacillus coli is delivered Chinese typical culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province, its deposit number is: CCTCCNO:M209303.
The expression and purification of embodiment 2 haemophilus parasuis immunogenic protein gene pilA in e. coli bl21 reaches the detection to the bloodthirsty infection protection of secondary pig
1. expression and the purification of haemophilus parasuis immunogenic protein gene pilA in e. coli bl21
For great expression pilA albumen, the applicant is transformed into the above-mentioned recombinant expression plasmid pET28a/Hps-pilA that carries its coded sequence in the e. coli bl21 (escherichia coli are available from Tiangen company), has obtained reorganization bacterium BL21/pET28a/Hps-pilA.This recombinant bacterial strain BL21/pET28a/Hps-pilA is inoculated in (additional final concentration is 50 μ g/mL kanamycin) in the 5mL LB fluid medium, the activation of spending the night.Be forwarded in the 50mL LB fluid medium with 1: 100 (v/v), place 37 ℃ of shaking tables to be cultured to OD
600After about 0.5-0.8, the isopropyl-B-D-thiogalactoside (being IPTG, available from sigma company) that adds 1.0mmol/L is in 37 ℃ of inducing culture 3h.The 3h inducing culture thing of above-mentioned 50mL reorganization bacterium BL21/pET28a/Hps-pilA is collected thalline through the centrifugal 30s of 12000rpm; Utilize ultrasound wave (technical parameter: power 400W; Broken 30s, intermittently 30s) the centrifugal 15min of 12000rpm behind the cell breakage is got supernatant.Then supernatant is crossed Ni-IDA affinity column (His nickel post) (available from Novagen company) this differential protein PilA (concrete purification step carries out according to the test kit operating instruction) that purifies.Final purified product is carried out the SDS-PAGE electrophoresis detection; The result is as shown in Figure 2; Albumen of purifying and the comparison of molecular weight of albumen standard; The estimation molecular weight is 15kDa, and is identical basically with the PilA molecular weight of albumen size of estimating, proves that haemophilus parasuis immunogenic protein pilA of the present invention has obtained successful expression in e. coli bl21.
2 pairs of haemophilus parasuis immunogenic proteins carry out the piglet antibody horizontal evaluation of immunity back
In order PilA albumen to be carried out the antibody horizontal evaluation of immunity back, the applicant is with the albumen of PilA and other 4 haemophilus parasuises, and inactivated vaccine and adjuvant contrast and carry out the piglet antibody horizontal evaluation of immunity back.Piglet is divided into 7 groups at random, every group of 3 pigs.The 1st group every intramuscular injection haemophilus parasuis inactivated vaccine 25 μ g (mixing) with the equal-volume aluminum hydroxide adjuvant.The recombiant protein of the 2nd～6 group of each haemophilus parasuis of injection (adding the equal-volume aluminum hydroxide adjuvant); The 7th group is matched group, the injection aluminum hydroxide adjuvant.The 1st time, every 200 μ g albumen, accumulated dose 3ml/, about 2 all collare venous blood collections (approximately 1ml) carry out ELIAS and detect.The 2nd time, dosage is 500 μ g/3ml/, carries out ELIAS after 1 week and detects.The 3rd time, dosage is 500 μ g/3ml/.0 week was immunity for the first time, and 2 weeks were immunity for the second time, and 4 weeks were immunity for the third time; Carry out blood sampling the 0th, 1,3,5 weeks and detected (Fig. 3).The result show PilA albumen two exempt from after, antibody horizontal has just reached level preferably.
3. the haemophilus parasuis immunogenic protein is to the detection of the bloodthirsty infection protection of secondary pig
The applicant is with the albumen of PilA and other 4 haemophilus parasuises; Inactivated vaccine and adjuvant contrast are carried out three and are exempted to carry out infection experiment with the haemophilus parasuis isolated strains after 7 days; Haemophilus parasuis SH0165 strain is through 21 pigs of intratracheal injection, and every pig is injected 3ml bacterium liquid 2 * 10
10/ only.The injection back was observed 6 days continuously.With purified proteins DnaK, GidA, PilA, RffA and SiaB immunity piglet, counteracting toxic substances is estimated the immune protective efficiency of recombinant expression protein.The protection power rate that causes death is as shown in Figure 4, and the deadly protective rate of inactivated vaccine and PilA protein immunization group is 100%, and the deadly protective rate of GidA albumen and adjuvant immunity group is 0%.
Estimate through scoring, the result is as shown in table 1.Wherein the PilA protein immunization is to negative control significant difference (p≤0.01), and the PilA protein immunization is to inactivated vaccine immunity difference not significantly (p＞0.05).
Table 1 haemophilus parasuis immunogenic protein is to the evaluation and test of the bloodthirsty infection protection of secondary pig
Sequence table
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att gag tta atg atc gtg atc gcc atc att gcc att tta gca aca ata 96
Ile Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile
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Leu Leu Ser Ala Ser Ala Ser Tyr Lys Ser Asp Val Glu Ile Cys Ile
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tat aac gta ggg gat aaa aaa atc tcg gaa tgt agt tca ggt aaa aat 240
Tyr Asn Val Gly Asp Lys Lys Ile Ser Glu Cys Ser Ser Gly Lys Asn
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ggc gtt cga gca gat aaa tcc gat gtc cct gac gcg aaa tac ctt aaa 288
Gly Val Arg Ala Asp Lys Ser Asp Val Pro Asp Ala Lys Tyr Leu Lys
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tct att tcc gtc gct tcg ggg gta atc acc gtg gca ggt aaa ggc aat 336
Ser Ile Ser Val Ala Ser Gly Val Ile Thr Val Ala Gly Lys Gly Asn
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att gat ggc tac ggc tac aca atg act ccg aaa ttc gct aat aac aac 384
Ile Asp Gly Tyr Gly Tyr Thr Met Thr Pro Lys Phe Ala Asn Asn Asn
115 120 125
atc act tgg gca aca agc tgt caa ggg gct gac att agt ttg ttc cct 432
Ile Thr Trp Ala Thr Ser Cys Gln Gly Ala Asp Ile Ser Leu Phe Pro
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20 25 30
Ala Ile Pro Ser Tyr Thr Ser Tyr Thr Gln Lys Ala Ala Leu Ser Glu
35 40 45
Leu Leu Ser Ala Ser Ala Ser Tyr Lys Ser Asp Val Glu Ile Cys Ile
50 55 60
Tyr Asn Val Gly Asp Lys LysIle Ser Glu Cys Ser Ser Gly Lys Asn
65 70 75 80
Gly Val Arg Ala Asp Lys Ser Asp Val Pro Asp Ala Lys Tyr Leu Lys
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Ser Ile Ser Val Ala Ser Gly Val Ile Thr Val Ala Gly Lys Gly Asn
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Ile Asp Gly Tyr Gly Tyr Thr Met Thr Pro Lys Phe Ala Asn Asn Asn
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Ile Thr Trp Ala Thr Ser Cys Gln Gly Ala Asp Ile Ser Leu Phe Pro
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Glu Asn Phe Cys Arg Gln
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Claims (1)
1. recombination bacillus coli (Escherichia coli) DH5 α/Hps-pilA of the immunogenic protein gene of haemophilus parasuis is expressed in a strain, is deposited in Chinese typical culture collection center (CCTCC), and deposit number is CCTCC NO:M209303.
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| CN102220272B (en) * | 2011-06-01 | 2013-06-19 | 武汉科前动物生物制品有限责任公司 | Method for high density culture of haemophilus parasuis for preparing vaccines |
| CN102864157B (en) * | 2011-07-05 | 2014-03-05 | 华中农业大学 | Immune protective antigen of haemophilus parasuis |
| CN103446581B (en) * | 2013-09-10 | 2015-06-17 | 中国农业科学院哈尔滨兽医研究所 | Method for preparing bacterial ghost vaccine of haemophilus parasuis as well as product and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1942204A (en) * | 2004-04-26 | 2007-04-04 | 株式会社中央疫苗研究所 | Inactivated mixed vaccine for porcine respiratory disease and the method of manufacturing thereof |
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| CN1942204A (en) * | 2004-04-26 | 2007-04-04 | 株式会社中央疫苗研究所 | Inactivated mixed vaccine for porcine respiratory disease and the method of manufacturing thereof |
Non-Patent Citations (3)
| Title |
|---|
| Qiyun Xie et al..Transcriptional responses of Haemophilus parasuis to iron-restriction stress in vitro.《Biometals》.2009,第22卷(第6期),907-916. * |
| 程安春等.鸭源致病性大肠杆菌Ⅰ型菌毛pilA 基因的原核表达及重组蛋白对强毒攻击的免疫保护作用.《生物工程学报》.2007,第23卷(第3期),440-445. * |
| 韦昭玉等.副猪嗜血杆菌的分子生物学研究进展.《韶关学院学报·自然科学》.2009,第30卷(第6期),74-79. * |
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