CN104962572A - Preparation method for aeromonas hydrophila outer membrane protein gene prokaryotic expression protein - Google Patents
Preparation method for aeromonas hydrophila outer membrane protein gene prokaryotic expression protein Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a preparation method for aeromonas hydrophila outer membrane protein gene prokaryotic expression protein. The method comprises the following steps that the protein clones an outer membrane protein gene segment into a prokaryotic expression vector pET-30a to obtain a recombinant expression vector, the recombinant expression vector is converted into Escherichia coli to obtain recombinant Escherichia coli, the recombinant Escherichia coli is inoculated to an Amp-resistant LB plate to be cultured for OD600 for 0.6 h, IPTG is added to reach the final concentration being 1.0 mmol/l for inducible expression, and the aeromonas hydrophila outer membrane protein gene prokaryotic expression protein is obtained after purification is performed. According to the method, the aeromonas hydrophila outer membrane protein gene prokaryotic expression protein is successfully constructed, the aeromonas hydrophila outer membrane protein gene prokaryotic expression protein can be used as a vaccine capable of protecting fish, the immune effects of the aeromonas hydrophila outer membrane protein gene prokaryotic expression protein pompA for channel catfish under different FIA effects are compared and researched, and a foundation is laid for further researching the pompA and researching whether the pompA can be used as the candidate component of the genetic engineering subunit vaccine or not.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of preparation method of Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen.
Background technology
Aeromonas hydrophila (Aeromonashydrophila) is extensively present in natural fresh water, dirty water and soil, by water and the related products infection mankind of pollution.Clinical symptom shows as gastro-enteritis, Skin and soft tissue infection, is also one of comparatively serious pathogenic bacteria of freshwater aquiculture.The hueppe's disease of fish, ponding, ulcer, asymptomatic septicemia and ophthalmoptosis etc. can be caused.This bacterium has various serotype, with the difference of region and host and difference to some extent, limits the use range of inactivated vaccine.The key of head it off is the common protective antigen finding out Aeromonas hydrophila, to prepare vaccine different serotypes bacterial strain all being had to provide protection.Outer membrane protein (outer membrane protein, OMP) is the main component of gram-negative bacteria cell wall.It is embedded in the centre of bacterial outer membrane, plays an important role in maintenance bacterial outer membrane structure, substance transportation.Have many results of study to show, the immunogenicity of the major outer membrane protein of bacterium is very strong, is important protective antigen, and ompA albumen race is main component in Gram-negative bacteria adventitia and is conservative, and its Main Function is maintain adventitia complete.Its form of Bacterial variation strain lacking ompA and mureinlipoprotein is spherical, and cytolemma is also unstable.Healthy animal has the opposing invasion and attack of pathogenic micro-organism and the ability of infection, body can rely on the barrier structures such as skin, mucous membrane, blood brain to stop entering of pathogenic micro-organism, also by the antimicrobial material in tissue and body fluid as complement, N,O-Diacetylmuramidase, Interferon, rabbit etc. carry out antibacterial, sterilization or bacteriolyze, this series of immune response of body limits or has resisted copying of bacterium.Therefore, the defence of host must be escaped or adapt to the pathogenic effects of pathogenic bacteria, and much research confirms, and the OMP of some bacterium plays an important role in bacterium pathogenic course.Analyze the colon bacillus OMP causing fowl septicemia with SDS-PAGE, find that fowl colon bacillus is non-and cause a disease or the very high LD of performance
50bacterial strain all lack 2 kinds of principle protein subunit of 40.7 and the 28.8KD only just had in pathogenic strains; illustrate that these 2 kinds of albumen may work in the pathogenic course of fowl septicemia; therefore, utilizing the OMP of pathogenic bacterium to do immunogen can make fish from the cross-protection produced different serotypes strain infection.
The nonspecific defense mechanism of fish can be divided into humoral defense and cytophylaxis two kinds of modes, and these two kinds of modes are all congenital formation, do not have specific performance, act on all exotic disease pathogenic microorganisms, also without heredity and memory.Wherein N,O-Diacetylmuramidase, complement and scavenger cell respiratory burst activity evaluates the important indicator of fish non-specific immunity defence.
As can be seen here, Outer Membrane Protein of Aeromonas Hydrophila gene can be utilized to carry out prokaryotic expression thus prepare a kind of albumen, making it can be used as vaccine fishing gear being had to provide protection, become the technical barrier that those skilled in the art are urgently to be resolved hurrily.
Summary of the invention
The present invention, in order to solve the problems of the technologies described above, provides a kind of preparation method of Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen.
Technical scheme of the present invention comprises:
A kind of Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen, described Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic protein amino acid sequence is SEQ ID NO.1, and described Outer Membrane Protein of Aeromonas Hydrophila gene nucleotide series is SEQID NO.2.
An application for Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen, described Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen can be used as antigen and uses in the vaccine preparation of preparation prevention Aeromonas hydrophila.
The vaccine preparation of described prevention Aeromonas hydrophila also comprises not formula Freund's incomplete adjuvant.
Described not formula Freund's incomplete adjuvant is 1:1 with described Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen volume ratio.
A kind of application of Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen, the preparation method of the vaccine preparation of described prevention Aeromonas hydrophila comprises the following steps: in asepsis injector, add the Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen prepared, in described asepsis injector, isopyknic not formula Freund's incomplete adjuvant is added with pipettor, after emulsification 5min, repeatedly advance with syringe, until aqueous phase and oil phase melt completely to together with emulsification complete, form water in oil oyster white drop, namely make vaccine preparation.
A preparation method for Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen, comprises the following steps:
Step one: the clone of Aeromonas hydrophila extracting genome DNA and Outer Membrane Protein of Aeromonas Hydrophila gene; Genomic dna is extracted from Aeromonas hydrophila strain isolated, according to Outer Membrane Protein of Aeromonas Hydrophila gene order design Auele Specific Primer, carry out pcr amplification reaction, by after the pcr amplification product purifying that obtains, pMD18-T carrier is cloned into the ratio of the ratio 1:3 of carrier and Insert Fragment amount of substance, containing picking positive colony on the LB flat board of penbritin, after double digestion and PCR qualification, obtain positive colony and extract plasmid DNA;
Step 2: expression plasmid builds: the described positive colony correct to order-checking extracts plasmid DNA and carry out BamHI, Hand III enzyme is cut, with identical restriction endonuclease, expression plasmid pET-30a enzyme is cut simultaneously, the goal gene fragment that glue purification recovery enzyme is cut and pET-30a carrier segments, connect reaction, obtain recombinant plasmid pET-32a (+)-pompA, by described recombinant plasmid transformed to competent escherichia coli cell, coating LB is dull and stereotyped, the single bacterium colony of picking, be inoculated in LB substratum and cultivate, extracting plasmid, qualification is cut through enzyme, the positive recombination bacillus coli comprising recombinant plasmid pET-32a (+)-pompA is obtained after the qualification of PCR reacting positive,
Step 3: the expression and purity of Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen: by described positive recombination bacillus coli streak inoculation in the LB slat chain conveyor of Amp resistance to OD
600when reaching 0.6, adding IPTG to final concentration is that 1.0mmol/L carries out abduction delivering, and the bacterium liquid of abduction delivering is carried out ultrasonic disruption and qualification, namely obtains Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen after washing, dissolving, filtration.
Described Auele Specific Primer is: upstream primer: 5 '-CCATGGGCATGTGTTTAC-3 '; Downstream primer: 5 '-GGATCCGACTGGTAGA-3 ', upstream primer sequence is SEQ ID NO.3, and downstream primer sequence is SEQ IDNO.4, adopts NcoI enzyme to cut described upstream primer, adopts BamH I enzyme to cut described downstream primer.
The system of described pcr amplification reaction is 25 μ L:2 × Taq Master Mix 12.5 μ l, DNA profiling 1 μ l, and upstream primer and each 1.0 μ l of downstream primer, add ddH
2o to 25 μ l; The condition of described pcr amplification reaction: 95 DEG C of denaturation 5min, 94 DEG C of 50s, 55 DEG C of 30s, 72 DEG C of 50s, totally 30 circulations, last 72 DEG C extend 10min, get 5 μ l reaction product and carry out electrophoresis with 1% sepharose, and gel imaging system detects amplification.
In described step 2, the method for extracting plasmid is alkaline lysis.
In described step 3 by described positive recombination bacillus coli streak inoculation in the method for the LB slat chain conveyor of Amp resistance be: 37 DEG C of overnight incubation, next day, picking list colony inoculation was in 10mL LB nutrient solution, 200rpm shaking culture is spent the night, be inoculated in 5mL LB nutrient solution in the ratio of 1:50 by the bacterium liquid of incubated overnight, thermal agitation is cultured to OD
600reach 0.6.
In described step 3, the process of abduction delivering is: after induction 4h, get 1mL bacterium liquid 4 DEG C, the centrifugal 1min of 12000rpm, supernatant discarded; Add the Tris-HCl damping fluid of 40 μ l and 5 × SDS sample-loading buffer of 10 μ l in precipitation, 100 DEG C of heating in water bath sex change 10min, then carry out 12%SDS-PAGE analysis.
The process of wash in described step 3, dissolve, filtering is: the precipitation of the bacterium liquid of the described abduction delivering after ultrasonic disruption and qualification is washed with the inclusion body washings containing 2M urea, after washing, the centrifugal 10min of 8000r/min removes supernatant, continuous 3 times, each 5min; Finally with the buffer solution recombinant protein containing 6M Guanidinium hydrochloride, be dissolved to limpid, by the solution after dissolving, with the membrane filtration in 0.45 μm of aperture.
Beneficial effect of the present invention comprises: the present invention chooses Outer Membrane Protein of Aeromonas Hydrophila ompA gene and carries out expressing and analyzing its immunogenicity in intestinal bacteria, a kind of preparation method of Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen is provided, and to combine with not formula Freund's incomplete adjuvant (FIA), the comparative studies immune effect of Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen pompA to channel catfish under different FIA effect.For studying ompA further and can laying the foundation as the Candidate components of genetic engineering subunit vaccine.
Accompanying drawing explanation
Figure 1 shows that pcr amplification result figure of the present invention.
Figure 2 shows that T cloned plasmids pMD19-T-pompA enzyme of the present invention cuts qualification and PCR qualification figure.
Figure 3 shows that recombinant expression plasmid pET-32a (+)-pompA enzyme of the present invention cuts qualification and PCR qualification figure.
Figure 4 shows that the present invention recombinates the expression analysis figure of pompA.
What Figure 5 shows that the present invention prevents the vaccine preparation of Aeromonas hydrophila on serum lysozyme level affects comparison diagram.
Figure 6 shows that the present invention prevents the vaccine preparation of Aeromonas hydrophila to serum ACH50 effect of vigor comparison diagram.
Figure 7 shows that treatment of different temperature serum bactericidal activity comparison diagram of the present invention.
Figure 8 shows that the present invention prevents the vaccine preparation of Aeromonas hydrophila active to head-kidney scavenger cell respiratory burst.
Figure 9 shows that the present invention prevents the vaccine preparation of Aeromonas hydrophila to serum conditioning phagocytosis comparison diagram.
What Figure 10 shows that the present invention prevents the vaccine preparation of Aeromonas hydrophila on channel catfish antibody horizontal affects comparison diagram.
Figure 11 shows that Aeromonas hydrophila of the present invention attacks the accumulative death toll spirogram of malicious channel catfish.
Figure 12 shows that Aeromonas hydrophila of the present invention attacks the tissue bacterial counting diagram of the rear channel catfish of poison.
Figure 13 shows that control group pathological study figure of the present invention; Figure 13 A control group renal interstitial 40 times of enlarged views; Figure 13 B immune group renal interstitial 40 times of enlarged views; Figure 13 C control group liver cell 40 times of enlarged views; Figure 13 D immune group liver cell 40 times of enlarged views; Figure 13 E control group meningeal tissue 40 times of enlarged views; Figure 13 F immune group meningeal tissue 40 times of enlarged views.
Embodiment
Hereafter will describe the specific embodiment of the invention in detail in conjunction with concrete accompanying drawing.It should be noted that the combination of technical characteristic or the technical characteristic described in following embodiment should not be considered to isolated, they can mutually be combined thus be reached better technique effect.
PompA+FIA described in ensuing disclosure is the vaccine preparation of prevention Aeromonas hydrophila of the present invention, and described pompA is Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen of the present invention.
Aeromonas hydrophila strain isolated used in the present invention is by genetic engineering procedure, and the Aeromonas hydrophila strain of isolating in the present invention can be separated in repetitive operation.
The separation method of Aeromonas hydrophila strain isolated comprises: get the sick fish that an infection has Aeromonas hydrophila, get disease fish liver under aseptic condition, nephridial tissue line is planted on General nutrition agar plate and Rabbit blood nutrition agar plates, cultivate 18hr, isolate pathogenic bacteria for 37 DEG C.
The authentication method of Aeromonas hydrophila strain isolated: according to morphological feature and the physio-biochemical characteristics of isolated strains, identifies with reference to the characteristic of Aeromonas in " uncle Jie Shi Systems Bacterial identification handbook ".
1, morphological observation: observe and get disease fish hepatic tissue printingout and pure growth smear, fix, microscopy after gramstaining.
The morphological specificity of isolated strains: isolated strains is Gram-negative tyrothricin, the blunt circle in two ends, without brood cell, is dispersed in or arranges in pairs, size 0.4 ~ 0.6 μm of X 1.0 ~ 2.2 μm, consistent with dying sick fish hepatic tissue printingout gram stain microscopy result.In smooth, circular, moistening, protuberance, neat in edge faint yellow bacterium colony on General nutrition agar plate.In strong β type haemolysis on Rabbit blood nutrition agar plates.Well-grown in nutrient broth, uniformity.
2, Physiology and biochemistry qualification: the pure growth being stored in ordinary nutrient agar slant is inoculated in respectively different bacteria bio trace assessors (sky, Hangzhou and microorganism reagent company limited produce), carries out biochemical characteristic qualification; According to the characteristic of isolated strains, namely dynamic, oxydase, by catalase-positive, breathe fermentating metabolism, fermentation grape sugar, fructose, nonfermented inositol, Pentitol, melampyrin, wood sugar and raffinose, hydrolyzed starch, polychrom, liquefy gelatin, not decomposing urea, reduction nitrate projects, meet the characteristic of Aeromonas hydrophila.
3, animal experiment: pure growth is inoculated in ordinary broth 37 DEG C and cultivates 18hr, get meat soup bacterium liquid 0.2ml abdominal injection 18 ~ 20 grams of small white mouses 4, separately establishes 2 to contrast the aseptic meat soup of injection, detects the virulence of isolated strains with minimum lethal dose.All dead within 48hr after animal experiment small white mouse inoculation meat soup bacterium liquid, control group is observed still normal to 15 days.Isolated strains is 2.0 hundred million bacteriums to the MLD of small white mouse.
4, isolated strains is to the susceptibility of antibacterials: adopt conventional paper disk method to carry out, and put 37 DEG C and cultivate 18hr, inhibition zone size is taken the mean.Drug sensitive test paper is purchased from Beijing the Temple of Heaven Pharmaceutical Biotechnology development company.
The sensitivity testing of isolated strains to antibacterials the results are shown in Table 1.
Table 1 isolated strains is to the susceptibility of antibacterials
According to morphological feature and the physio-biochemical characteristics of isolated strains, with reference to the characteristic of Aeromonas in " uncle Jie Shi Systems Bacterial identification handbook ", isolate is decided to be Aeromonas hydrophila.Although some difference of some biochemical characteristic of isolated strains, the performance of its main characteristic is consistent, and this does not affect the qualification result of this bacterium.
Embodiment 1
A kind of Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen, described Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic protein amino acid sequence is SEQ ID NO.1, and described Outer Membrane Protein of Aeromonas Hydrophila gene nucleotide series is SEQID NO.2.Described Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen can be used as antigen and uses in the vaccine preparation of preparation prevention Aeromonas hydrophila.The vaccine preparation of described prevention Aeromonas hydrophila also comprises not formula Freund's incomplete adjuvant.Described not formula Freund's incomplete adjuvant is 1:1 with described Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen volume ratio.
A preparation method for Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen, comprises the following steps:
Step one: the clone of Aeromonas hydrophila extracting genome DNA and Outer Membrane Protein of Aeromonas Hydrophila gene; Extract genomic dna from Aeromonas hydrophila strain isolated, according to Outer Membrane Protein of Aeromonas Hydrophila gene order design Auele Specific Primer, described Auele Specific Primer is: upstream primer: 5 '-CCATGGGCATGTGTTTAC-3 '; Downstream primer: 5 '-GGATCCGACTGGTAGA-3 ', upstream primer sequence is SEQ ID NO.3, and downstream primer sequence is SEQ IDNO.4, adopts NcoI enzyme to cut described upstream primer, adopts BamH I enzyme to cut described downstream primer.Carry out pcr amplification reaction, the system of described pcr amplification reaction is 25 μ L:2 × Taq Master Mix 12.5 μ l, DNA profiling 1 μ l, and upstream primer and each 1.0 μ l of downstream primer, add ddH
2o to 25 μ l; The condition of described pcr amplification reaction: 95 DEG C of denaturation 5min, 94 DEG C of 50s, 55 DEG C of 30s, 72 DEG C of 50s, totally 30 circulations, last 72 DEG C extend 10min, get 5 μ l reaction product and carry out electrophoresis with 1% sepharose, and gel imaging system detects amplification.By after the pcr amplification product purifying that obtains, pMD18-T carrier is cloned into the ratio of the ratio 1:3 of carrier and Insert Fragment amount of substance, containing picking positive colony on the LB flat board of penbritin, after double digestion and PCR qualification, obtain positive colony and extract plasmid DNA;
Step 2: expression plasmid builds: the described positive colony correct to order-checking extracts plasmid DNA and carry out BamHI, Hand III enzyme is cut, with identical restriction endonuclease, expression plasmid pET-30a enzyme is cut simultaneously, the goal gene fragment that glue purification recovery enzyme is cut and pET-30a carrier segments, connect reaction, obtain recombinant plasmid pET-32a (+)-pompA, by described recombinant plasmid transformed to competent escherichia coli cell, coating LB is dull and stereotyped, the single bacterium colony of picking, be inoculated in LB substratum and cultivate, alkaline lysis extracting plasmid, qualification is cut through enzyme, the positive recombination bacillus coli comprising recombinant plasmid pET-32a (+)-pompA is obtained after the qualification of PCR reacting positive,
Step 3: the expression and purity of Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen: by described positive recombination bacillus coli streak inoculation in the LB slat chain conveyor of Amp resistance, i.e. 37 DEG C of overnight incubation, next day, picking list colony inoculation was in 10mL LB nutrient solution, 200rpm shaking culture is spent the night, be inoculated in 5mL LB nutrient solution in the ratio of 1:50 by the bacterium liquid of incubated overnight, thermal agitation is cultured to OD
600reach 0.6.Adding IPTG to final concentration is that 1.0mmol/L carries out abduction delivering, and the process of abduction delivering, for after induction 4h, gets 1mL bacterium liquid 4 DEG C, the centrifugal 1min of 12000rpm, supernatant discarded; Add the Tris-HCl damping fluid of 40 μ l and 5 × SDS sample-loading buffer of 10 μ l in precipitation, 100 DEG C of heating in water bath sex change 10min, then carry out 12%SDS-PAGE analysis.The bacterium liquid of abduction delivering is carried out ultrasonic disruption and qualification, the precipitation of the bacterium liquid of the described abduction delivering after ultrasonic disruption and qualification is washed with the inclusion body washings containing 2M urea, after washing, the centrifugal 10min of 8000r/min removes supernatant, continuous 3 times, about 5min at every turn; Finally with the buffer solution recombinant protein containing 6M Guanidinium hydrochloride, be dissolved to limpid, by the solution after dissolving, namely obtain Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen with after the membrane filtration in 0.45 μm of aperture.
A kind of application of Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen, the preparation method of the vaccine preparation of described prevention Aeromonas hydrophila comprises the following steps: in asepsis injector, add the Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen prepared, in described asepsis injector, isopyknic not formula Freund's incomplete adjuvant is added with pipettor, after emulsification 5min, repeatedly advance with syringe, until aqueous phase and oil phase melt completely to together with emulsification complete, form water in oil oyster white drop, namely make vaccine preparation.
The amplification of embodiment 2 Aeromonas hydrophila ompA gene, T cloning and identification
With Aeromonas hydrophila PDK06 pnca gene group DNA for template, according to designed PCR primer, as shown in Figure 1, go out through pcr amplification the object segment that is positioned at 1000bp-1100bp interval.PCR primer reclaims after purifying through glue, be connected with pMD19-T carrier and obtain recombinant plasmid pMD19-T-ompA, recombinant plasmid pMD19-T-ompA sequencing result is shown, the ompA gene of Aeromonas hydrophila PDK06 strain is by the Complete Open reading frame (ORF) of 1035 based compositions, a length of encoding is 275 amino acid whose albumen, and recombinant plasmid is cut with PCR qualification all correct through enzyme.
The pcr amplification of embodiment 3 Aeromonas hydrophila ompA gene, the structure of T Cloning and Expression carrier
Reclaim rear clone to pMD19-T carrier through glue, then carry out single double digestion qualification and order-checking qualification.Single double digestion qualification result shows as shown in Figure 2: M1 is DNA marker DL2000,1 is pompA gene PCR amplified production, 2 be pMD19-T-pompA through BamH I and Hind III double digestion product, 3 be pMD19-T-pompA through BamH I single endonuclease digestion product, M2 is DNA marker 10000; Single endonuclease digestion linearizing recombinant vectors is about about 3600bp, and two bands appear in double digestion, respectively about 1000bp and 2500bp, and sequencing result and design of primers reference sequences are 100% with section similarity.As shown in Figure 3: M1, DNA Marker (DL2,000); 1, pcr amplification product; 2, pET-32a (+)-pompA cuts through BamHI and HindIII enzyme; 3, pET-32a (+)-pompA cuts through BamHI enzyme; 4, pET-32a (+) is through BamHI and Hind III digestion; 5, pET-32a (+) cuts through BamHI enzyme; As shown in Figure 3, enzyme is cut after object fragment glue reclaims and be connected to expression vector pET-32a (+), consistent with expected results through single double digestion display.
Embodiment 4 recombinant plasmid pET-32a (+)-pompA abduction delivering and purifying
Correct recombinant plasmid pET-32a (+)-pompA of qualification is converted in BL21 (DE3), the bacterium liquid of incubated overnight is inoculated in 5mLLB nutrient solution in the ratio of 1:50, adding IPTG to final concentration is 1.0mmol/L, carry out SDS-PAGE inspection after induction, occur the band of about 41.4KDa size.Thalline after induction, through multigelation, is separated upper cleer and peaceful precipitation after ultrasonic disruption, find that expressing recombinant protein exists with inclusion bodies.As shown in Figure 4: M, protein marker; 1, the BL21-pET-32a (+) do not induced; BL21-pET-32a (+) after 2, IPTG induction; BL21-pET-32a (+)-pompA supernatant after 3, IPTG induction; BL21-pET-32a (+)-pompA after 4, IPTG induction precipitates; 5, the pompA after purifying; As shown in Figure 4, purifying protein concentrates after gradient urea element renaturation, and protein concentrate nucleic acid-protein instrument measures concentration and reaches 2.5mg/mL.
Embodiment 5 salivary lysozyme detects
1, serum lysozyme level detects
The impact of vaccine preparation on channel catfish lysozyme activity of prevention Aeromonas hydrophila of the present invention is shown in 5.Known as shown in Figure 5, FIA vaccine group antalzyme activity all pole is significantly higher than control group and without Adjuvanted vaccines group antalzyme activity (p<0.01), is all significantly higher than control group antalzyme activity (p<0.05) without Adjuvanted vaccines group antalzyme activity.Not significantly (p>0.05), it is consistent that the antigen slow releasing of parcel can stimulate body to continue to produce the result of immunne response by this result and FIA for 4th week to the 8th week immune group antalzyme activity difference.Learn according to above result, the vaccine preparation group of prevention Aeromonas hydrophila of the present invention can continue to stimulate channel catfish to produce the antalzyme activity of higher level; And simple subunit vaccine pompA also obviously enough can improve the antalzyme activity of channel catfish compared with control group.
2, ACH50 Activity determination
Fig. 6 is shown in the impact of vaccine preparation on channel catfish ACH50 vigor of prevention Aeromonas hydrophila.Known as shown in Figure 6, vaccine group ACH50 vigor all pole is significantly higher than control group A CH50 vigor (p<0.01), FIA vaccine group ACH50 vigor is also significantly higher than without Adjuvanted vaccines group (p<0.05), 4th, 6 and 8 weeks, each group of ACH50 vigor is all more stable, test group slightly improves, and demonstrating FIA can stimulate body to continue to produce immunne response by the antigen slow releasing of parcel.Learn according to above result, the vaccine preparation group of prevention Aeromonas hydrophila can continue to stimulate channel catfish to produce the ACH50 vigor of higher level, and simple subunit vaccine pompA also significantly enough can improve the ACH50 vigor of channel catfish compared with control group.
3, serum sterilizing is lived in going and is detected
In order to measure the effect of complement and antibody in serum sterilizing, respectively by immune group and control group the 4th week serum through 56 DEG C of process, 100 DEG C of process and untreated, have detected the serum bactericidal activity of different group.Known as shown in Figure 7, the Survival probability of bacteria of the untreated serum of immune group is minimum, next is 56 DEG C of process serum and 100 DEG C of process serum Survival probability of bacterias, and difference extremely significantly (p<0.01), illustrates that complement in serum and antibody play an important role in serum sterilizing between each process.Untreated serum FIA+pompA group fungicidal activity is the strongest, but with pompA group difference remarkable (p>0.05); 56 DEG C of process serum FIA+pompA group fungicidal activities are the strongest, difference is organized extremely significantly (p<0.01) with other, and pompA group serum Survival probability of bacteria is higher than FIA+pompA group, significantly lower than control group (p>0.05); It is minimum that 100 DEG C of process serum remains FIA+pompA group Survival probability of bacteria, and significantly lower than control group and pompA group, and pompA group Survival probability of bacteria is close to control group, illustrates that in pompA group, antibody-mediated sterilization plays a major role.
4, respiratory burst Activity determination
Nitroblue tetrazolium (NBT) method is adopted to measure head-kidney scavenger cell respiratory burst active.Known as shown in Figure 8, between the FIA subunit vaccine group of mensuration three time points, head-kidney scavenger cell respiratory burst activity difference is not significantly (p>0.05), prevent the vaccine preparation group of Aeromonas hydrophila and pompA group and control group difference extremely remarkable (p<0.01), pompA group and control group significant difference (p<0.05), illustrate that FIA can strengthen channel catfish head-kidney scavenger cell respiratory burst vigor.
5, opsono-cytophagic test
4th week immune group and control group serum, through 56 DEG C of heat treated, with deactivation complement, determine the conditioning phagocytosis of specific antibody to Aeromonas hydrophila.Known as shown in Figure 9, the Aeromonas hydrophila of FIA+pompA group serum process is maximum by the quantity of channel catfish head-kidney macrophage phagocytic, next is followed successively by AHA+pompA group, PA+pompA group, pompA group and PBS group, and each group difference extremely significantly (p<0.01).
6, antibody horizontal detects
Determine immune group and control group the 3rd week to the 8th week antibody horizontal respectively.Known as shown in Figure 10, FIA vaccine group antibody horizontal reaches peak value on the 5th week, and pompA group the 4th week antibody horizontal reaches peak value.Within 3rd week to the 8th week, FIA+pompA group antibody horizontal is the highest, and pole is significantly higher than all the other each group (p<0.01); Within 3rd week to the 6th week, pompA group antibody horizontal is also significantly higher than control group (p<0.01) in pole; 7th, 8 weeks pompA group antibody horizontals and control group do not have significant difference (p>0.05).Illustrate that FIA can improve the antibody horizontal of subunit vaccine pompA, and antibody horizontal can be maintained in the long period.
7, death condition and premunition protection ratio
Known as shown in figure 11, after test fish attacks poison, 3d starts dead successively, and FIA+pompA group 5d starts death, and each group reaches the peak mortality phase at 8d to 10d respectively.The cumulative mortality of PBS, FIA, pompA and FIA+pompA group Continuous Observation 14d is respectively 93.33%, 93.33%, 50.00% and 13.33%.PompA and FIA+pompA group relative protection ratio is respectively 46.43% and 85.71%.
8, histoorgan bacterial count
Known as shown in figure 12, after attacking poison, 48h liver, kidney and spleen tissue bacterial count result show, and each group spleen and kidney bacterial number, all higher than liver, are bred the fastest in kidney and spleen tissue after infected by Aeromonas hydrophila channel catfish is described.In FIA+pompA group liver organization, bacteria content is minimum, and these two groups are organized bacteria content difference extremely remarkable (p<0.01) in liver organization with other, in pompA group liver organization, bacteria content and control group difference are not significantly (p>0.05); In FIA+pompA group renal tissue, bacteria content is minimum, and respectively organize significant difference (p<0.05) with other, and between other groups, in renal tissue, bacteria content difference is not all significantly (p>0.05); In FIA+pompA group spleen tissue, bacteria content is minimum.
9, pathological study
After attacking poison, 3d test fish starts death, and the spot that fades appears in dead fish body surface, and indivedual fin bar base portion has bleeding.Find liver enlargement after cuing open inspection, congested, matter is crisp; Kidney enlargement, has blutpunkte; Spleen enlargement, to darken; Meninx is congested; Other internal organs are without obvious pathology.Inoculate dead fish liver, kidney, spleen tissue in BHI flat board, growing single tip-like bacterium colony, dyeing as Gram-negative bacteria, through being accredited as Aeromonas hydrophila.
Each group of inspection test fish is cutd open respectively the 4th week, 6 weeks and 8 weeks, observe and find, 8th week FIA group and FIA+pompA group are still attached with a large amount of white " milky " adjuvant drop around channel catfish mesentery, but do not observe granuloma and tissue injury in injection site.
Pathological study finds: as shown in FIG. 13A, control group renal interstitial oedema, capillary injection, there is the necrosis region differed in size in many places, inside has inflammatory cell infiltration, and glomerular capillary is congested, renal cells degeneration necrosis, inflammatory cell infiltration around tube chamber; As shown in fig. 13 c, liver Cable Structure is unintelligible, occurs inflammatory cell in central vein tube chamber, tube chamber endotheliocytic swelling, part necrocytosis, swelling of liver cell, serious vacuolar degeneration, and part of hepatocytes is downright bad; As shown in figure 13e, meninx oedema thickens, and capillary vessel is seriously congested, and with inflammatory cell infiltration, neuronal cell degeneration necrosis.And immune group renal interstitial capillary injection, as shown in Figure 13 B, glomerular capillary is congested, endotheliocytic swelling, renal cells sex change, and stratum basale is exposed; Hepatic tissue clear in structure, slight hepatic cell sex change, part nuclear condensation, color burn, there is necrosis region in local; As illustrated in figure 13d, there is little cavity in part cell cytosol, vacuolar degeneration of hepatic cell around stove in slight hepatic cell sex change; As shown in Figure 13 F, meninx Mild edema, local capillary filling, glial cells hyperplasia, occurs addicted to neurological phenomena.
The present invention determines the impact on its nonspecific immune response after not formula Freund's incomplete adjuvant (FIA) geometric ratio mixing subunit vaccine pompA immunodotting Cha Wei Channel-catfish, result display FIA can significantly strengthen channel catfish lysozyme activity, ACH50 is active and head-kidney scavenger cell respiratory burst is active.Three salivary lysozymes the 4th week, the 6th week and the 8th week change difference in each group remarkable, wherein the lysozyme activity of FIA group and respiratory burst activity are all higher than PA group.
After specific immunity refers to that body is subject to the stimulation of foreign antigenic foreign matter, there is a series of immune response in the associated immune cells in body, to get rid of foreign antigenic foreign matter, keeps the relatively stable of organismic internal environment, have acquired character, specificity.Immunoglobulin (Ig) (immune globulin, Ig) is topmost medium in fish specific humoral immune response, mainly secretes in blood and the immunity of other body fluid conductors liquid with the antibody formation of solubility.Embodiment of the present invention result display pompA group antibody horizontal peak value appears at the 4th week, consistent with result of study before, and FIA group antibody horizontal reached peak value at the 5th week, there are differences with research before, this may have relation with immune programme for children and vaccine itself.A lot of people has carried out the research of vaccines for fish with oily adjuvant, and result shows it can make antigen slow releasing and continuous action, increases antigen to immune open-assembly time, thus the antibody horizontal that the maintenance of energy longer time is higher.The display of this result of study is cutd open inspection immune group channel catfish and is found, within the 8th week, still can see white " milky " drop at test fish intraperitoneal, and other groups has no vaccine or adjuvant.
The immunoglobulin (Ig) of fish is mainly IgM, when exotic antigen enters fish body inside, by the Presentation of antigen, B cell proliferation is made to be divided into plasmocyte, synthesis is secreting specificity antibody also, the Fab fragment of antibody can directly be combined with antigen, and the Fc receptors bind on Fc fragment energy and phagocytic cell surface, promote cytophagous engulfing.After the serum of different immune group is processed deactivation complements by 56 DEG C by this research, determine the conditioning phagocytosis of each group of process Serum Antibody mediation, result shows, the serum of FIA group has stronger opsonophagocytosis ability, be significantly higher than pompA group, the generation of the induction channel catfish potent antibodies of adjuvant energy higher level is described.
Premunition protection ratio (RPS) is the index evaluating vaccine effect most cogency.This result of study display subunit vaccine pompA only has medium immune protective effect, and premunition protection ratio is 46.43%, and after adding FIA, premunition protection ratio significantly increases, and FIA group premunition protection ratio is respectively 85.71%.
After attacking poison, bacterium is also one of important indicator evaluating immune effect of vaccine in the pathology damage degree of each histoorgan quantity and each histoorgan.After cause of disease enters body, arrive each histoorgan by blood circulation, and in this process, body immune system is excited, each immunocyte and immune factor participate in the removing to cause of disease jointly.The rear 48h of poison is attacked in the display of embodiment of the present invention result, bacterial number in each group of liver is starkly lower than the quantity of bacterium in spleen and kidney, this may be that Aeromonas hydrophila enters these two tissues more easily by phagocytic cell and blood circulation because kidney and spleen are important hematopoiesis and immune organ.Bacteria content in FIA group liver, kidney and spleen is all lower than other groups.All there is the symptoms such as serious sex change, necrosis and hyperemia is hemorrhage in simultaneously pathological study result display control group liver, kidney and brain, and the pathological change of FIA group respective organization is not obvious, illustrate that FIA can stimulate channel catfish immunity system, produce immunne response, strengthen the resistivity to Aeromonas hydrophila.
The present invention chooses Outer Membrane Protein of Aeromonas Hydrophila ompA gene and carries out expressing and analyzing its immunogenicity in intestinal bacteria, and to combine with not formula Freund's incomplete adjuvant (FIA), the comparative studies immune effect of Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen pompA to channel catfish under different FIA effect.For studying ompA further and can laying the foundation as the Candidate components of genetic engineering subunit vaccine.
Above-mentioned detailed description is the illustrating of possible embodiments for invention, and this embodiment is also not used to limit the scope of the claims of the present invention, does not allly depart from equivalence of the present invention and implements or change, and all should be contained in the scope of the claims of the present invention.
In addition, those skilled in the art also can make various amendments in other form and details, interpolation and replacement in the claims in the present invention scope of disclosure and spirit.Certainly, the changes such as these various amendments made according to the present invention's spirit, interpolation and replacement, all should be included within the present invention's scope required for protection.
Claims (7)
1. a preparation method for Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen, is characterized in that, comprises the following steps:
Step one: the clone of Aeromonas hydrophila extracting genome DNA and Outer Membrane Protein of Aeromonas Hydrophila gene: extract genomic dna from Aeromonas hydrophila strain isolated, according to Outer Membrane Protein of Aeromonas Hydrophila gene order design Auele Specific Primer, carry out pcr amplification reaction, by after the pcr amplification product purifying that obtains, pMD18-T carrier is cloned into the ratio of the ratio 1:3 of carrier and Insert Fragment amount of substance, picking positive colony on the LB flat board containing penbritin, after double digestion and PCR qualification, obtain positive colony and extract plasmid DNA;
Step 2: expression plasmid builds: the described positive colony correct to order-checking extracts plasmid DNA and carry out BamHI, Hand III enzyme is cut, with identical restriction endonuclease, expression plasmid pET-30a enzyme is cut simultaneously, the goal gene fragment that glue purification recovery enzyme is cut and pET-30a carrier segments, connect reaction, obtain recombinant plasmid pET-32a (+)-pompA, by described recombinant plasmid transformed to competent escherichia coli cell, coating LB is dull and stereotyped, the single bacterium colony of picking, be inoculated in LB substratum and cultivate, extracting plasmid, qualification is cut through enzyme, the positive recombination bacillus coli comprising recombinant plasmid pET-32a (+)-pompA is obtained after the qualification of PCR reacting positive,
Step 3: the expression and purity of Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen: by described positive recombination bacillus coli streak inoculation in the LB slat chain conveyor of Amp resistance to OD
600when reaching 0.6, adding IPTG to final concentration is that 1.0mmol/L carries out abduction delivering, and the bacterium liquid of abduction delivering is carried out ultrasonic disruption and qualification, namely obtains Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen after washing, dissolving, filtration.
2. the preparation method of a kind of Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen according to claim 1, it is characterized in that, described Auele Specific Primer is: upstream primer: 5 '-CCATGGGCATGTGTTTAC-3 '; Downstream primer: 5 '-GGATCCGACTGGTAGA-3 ', adopts NcoI enzyme to cut described upstream primer, adopts BamH I enzyme to cut described downstream primer.
3. the preparation method of a kind of Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen according to claim 2, it is characterized in that, the system of described pcr amplification reaction is 25 μ L:2 × Taq Master Mix 12.5 μ l, DNA profiling 1 μ l, upstream primer and each 1.0 μ l of downstream primer, add ddH
2o to 25 μ l; The condition of described pcr amplification reaction: 95 DEG C of denaturation 5min, 94 DEG C of 50s, 55 DEG C of 30s, 72 DEG C of 50s, totally 30 circulations, last 72 DEG C extend 10min, get 5 μ l reaction product and carry out electrophoresis with 1% sepharose, and gel imaging system detects amplification.
4. the preparation method of a kind of Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen according to claim 1, it is characterized in that, in described step 2, the method for extracting plasmid is alkaline lysis.
5. the preparation method of a kind of Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen according to claim 1, it is characterized in that, in described step 3 by described positive recombination bacillus coli streak inoculation in the method for the LB slat chain conveyor of Amp resistance be: 37 DEG C of overnight incubation, next day, picking list colony inoculation was in 10mL LB nutrient solution, 200rpm shaking culture is spent the night, be inoculated in 5mL LB nutrient solution in the ratio of 1:50 by the bacterium liquid of incubated overnight, thermal agitation is cultured to OD
600reach 0.6.
6. the preparation method of a kind of Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen according to claim 1, it is characterized in that, in described step 3, the process of abduction delivering is: after induction 4h, get 1mL bacterium liquid 4 DEG C, the centrifugal 1min of 12000rpm, supernatant discarded; Add the Tris-HCl damping fluid of 40 μ l and 5 × SDS sample-loading buffer of 10 μ l in precipitation, 100 DEG C of heating in water bath sex change 10min, then carry out 12%SDS-PAGE analysis.
7. according to the preparation method of a kind of Outer Membrane Protein of Aeromonas Hydrophila gene prokaryotic albumen according to claim 1, it is characterized in that, the process of wash in described step 3, dissolve, filtering is: the precipitation of the bacterium liquid of the described abduction delivering after ultrasonic disruption and qualification is washed with the inclusion body washings containing 2M urea, after washing, the centrifugal 10min of 8000r/min removes supernatant, continuous 3 times, each 5min; Finally with the buffer solution recombinant protein containing 6M Guanidinium hydrochloride, be dissolved to limpid, by the solution after dissolving, with the membrane filtration in 0.45 μm of aperture.
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