CN108330142B - Mermaid photorhabditis hemolysin Hly with immune protection effectchProtein - Google Patents

Abstract

The invention discloses mermaid photorhabditis hemolysin Hly with immune protection effect_chThe protein is prepared by constructing a recombinant vector pMD18T-hly_chThe recombinant expression vector pET32a-hly_chAnd prokaryotic expression hly_chGene preparation of recombinant Hly_chProtein, and Hly verified by SDS-PAGE analysis and Western blot_chProtein, and animal protection experiments to evaluate its protective power. Hly of photobacterium mermaides obtained in the present invention_chThe protein has good immune protection. Lays a foundation for developing mermaid photobacterium gene engineering subunit vaccine.

Description

A kind ofMermaid photorhabditis hemolysin Hly with immune protection effectchProtein

Technical Field

The invention relates to the technical field of biology, in particular to mermaid photorhabdus hemolysin Hly_chA method for preparing and identifying protein.

Background

Photobacterium damselae (Photobacterium damselae) is an important member in halophilic pathogenic bacteria, and diseased fish infected with the Photobacterium is clinically characterized by hemorrhagic septicemia, has rapid onset of disease and high mortality rate, and causes great economic loss to the marine economic fish culture industry. Humans and other mammals can also develop infections, a pathogenic bacteria with which humans-animals-fish are co-infected. The highly pathogenic photobacterium mermaides is known to carry usually about 150kb virulence plasmid encoding two key virulence factors, hemolysin Dly protein and HlyA_plThe protein, and the chromatin also carry the hemolysin Hly_chThe genes, these three hemolysins all have different degrees of influence on the virulence of bacteria. The hemolysin is one of important virulence factors of the photobacterium mermais, can act on lipid bilayers of red blood cells to form cavities of the cell membranes, change the permeability of the cell membranes and cause the rupture and dissolution of the red blood cells, thereby releasing more heme and providing more Fe for the heme³⁺Eventually leading to necrosis of tissues and organs of the mariculture animals and even death of infected animals.

In 11 months of 2015, turbots (Turbot) bred in a certain mariculture plant in Changli county of Qinhuang island are in a large amount of diseases, diseased fishes are manifested as body surface ulcers, fin bleeding, tail ulceration and severe ascites, the lethality rate is up to 80%, and the important etiological significance is highlighted. The subject group separated and identified mermaid photobacterium from ascites, and named MCCC 1K03226 by China Marine microorganism Collection center. In view of the high pathogenicity of photobacterium mermaid and the important role of hemolysin, the carrying condition of photobacterium mermaid hemolysin and the immune protection effect of hemolysin are researched to develop a genetic engineering subunit vaccine to finally achieve the aim of controlling the disease, and the photobacterium mermaid is a problem which needs to be solved by technical personnel in the field.

Disclosure of Invention

In view of the above, the invention provides a mermaid photorhabditis hemolysin Hly with immune protection effect_chThe protein is expressed and purified by a prokaryotic expression system, and the hemolysin Hly is analyzed_chImmunogenicity of the protein, and animal protection experiments are carried out. In order to achieve the purpose, the invention adopts the following technical scheme:

mermaid photorhabditis hemolysin Hly with immune protection effect_chThe protein is applied to the development of mermaid photobacterium gene engineering subunit vaccines.

Furthermore, the mermaid photorhabdus hemolysin Hly with immune protection function_chThe preparation method of the protein comprises the following specific steps

1) Recombinant vector pMD18T-hly_chConstruction of

Extracting genome DNA of photobacterium mermairei, wherein the strain preservation number of the photobacterium mermairei is MCCC 1K03226, the genome DNA is used as a template, and Hly is used_ch-F、Hly_ch-R is Hly of primer amplification removing signal peptide coding sequence_chGene, amplification of Hly by touchdown PCR_chFull length of gene, the primer Hly_ch-F、Hly_ch-the sequence of R is as follows:

Hly_ch-F：5'-GCGGATCCGACGTTATATCAATATTAAGCAG-3'; carrying a BamH I enzyme cutting site; SEQ ID NO: 10;

Hly_ch-R：5'-GCGTCGACTTACTGATTTATGGTTGATGGGTC-3'; carrying Sal I enzyme cutting site; SEQ ID NO: 11;

the hly to be obtained_chThe gene is connected with a T vector to obtain a recombinant vector pMD18T-hly_chAnd the recombinant vector pMD18T-hly_chSequencing and protein subcellular localization;

2) recombinant expression vector pET32a-hly_chConstruction of

The recombinant vector pMD18T-hly was separately digested with restriction enzymes BamH I and Sal I_chAnd expression vector pET32a (+)Performing double enzyme digestion, recovering enzyme digestion products by gel electrophoresis, mixing the enzyme digestion products in proportion, connecting the mixture for 2 to 3 hours at the temperature of between 22 and 25 ℃, transforming escherichia coli competent cells, inoculating the escherichia coli competent cells into an LB plate I, and culturing the escherichia coli competent cells overnight at the temperature of 37 ℃; picking single colony shake bacteria, and performing PCR identification by using a carrier universal primer; enlarging and culturing the positive colony, preserving the strain and extracting the plasmid pET32a-hly_ch；

3)hly_chProkaryotic expression of

The plasmid pET32a-hly was introduced_chThe transformed expression strain BL21 is spread on a second LB plate and cultured overnight at 37 ℃; picking the single colony of the second LB plate to shake bacteria overnight, sucking 1mL of bacterial liquid the next day, inoculating the bacterial liquid into 100mL of liquid LB culture medium, shaking the bacteria at 220r/min until OD is reached₆₀₀The value reaches 0.6-0.8, and IPTG is added to induce expression for 4-6 h.

Further, the recombinant vector pMD18T-hly in step 2)_chThe ratio of the product obtained by the restriction enzyme digestion of the expression vector pET32a (+) to the product recovered by the restriction enzyme digestion of the expression vector pET32a is 3: 1. Wherein, the recombinant vector pMD18T-hly_chThe ratio of the product obtained by the restriction enzyme digestion of the expression vector pET32a is too high or too low, which results in the recombinant expression vector pET32a-hly_chIt is difficult to construct successfully.

Further, the competent cell in step 2) is DH5 α.

Further, the first LB plate and the second LB plate in step 2) are solid LB plates containing ampicillin at a concentration of 45-55. mu.g/L. Among them, ampicillin concentration was too low or too high, which resulted in failure to screen positive colonies.

Further, the liquid LB in the step 3) is liquid LB containing ampicillin, and the concentration of the ampicillin is 45-55 mug/L. Wherein, if the concentration of ampicillin is too low, the screening pressure is not enough, the recombinant expression vector is easy to lose, and if the concentration of ampicillin is too high, the growth of bacteria is poor.

Further, the concentration of IPTG in step 3) is 1 mM. Wherein, when the concentration of IPTG is too high, the growth of bacteria is inhibited, and when the concentration is too low, the expression quantity of protein is insufficient.

Mermaid photorhabdus hemolysin Hly_chMethods for identifying proteins, including SDS-PAGE analysis and Western blot validation;

centrifuging the bacterial suspension, collecting thalli, washing for 3 times by PBS, then resuspending, adding a Loading Buffer, boiling and cracking, carrying out SDS-PAGE, and after electrophoresis is finished, carrying out gel staining and decoloring to observe a result; and simultaneously carrying out Western blot to verify the expression of the target protein.

Further, the concentration of the separation gel in the SDS-PAGE electrophoresis was 12%. Wherein, the acrylamide with the content of 12 percent is used, and the protein separation effect is better.

Further, in the Western blot method, the primary antibody is a monoclonal antibody with a His label, the concentration is 1:6000, and the secondary antibody is goat anti-mouse IgG-HRP, and the concentration is 1: 4000.

The mermaid photorhabdus hemolysin Hly_chProteins, the immunogenicity of which was assessed by ELISA; the method comprises the following steps:

induction of Hly_chExpressing protein, ultrasonic cracking thallus, centrifuging, taking supernatant and precipitate, performing SDS-PAGE, and determining Hly_chThe protein is expressed by inclusion body; purifying the protein by using a QIAGEN Ni-NTA column; and (3) coating the ELISA plate with 10 ng/hole protein, taking mouse immune serum as a primary antibody, taking an HRP-goat anti-rabbit secondary antibody as an enzyme-labeled antibody, performing ELISA (enzyme-linked immuno sorbent assay) determination, and determining the antibody titer.

The mermaid photorhabdus hemolysin Hly_chProteins whose immunoprotection was assessed by animal experiments;

the animal experiment evaluation method comprises the following steps: after the boosting, the bacteria are detoxified at two weeks, Balb/c mouse mermaid photobacterium MCCC 1K03226 is injected into the abdominal cavity, and the dosage is 5LD₅₀(i.e., 6X 10)⁷cfu), the number of dead mice was counted within one week after infection.

The invention discloses and provides mermaid photorhabdus hemolysin Hly_chThe preparation and identification method of the protein has at least the following advantages:

(1) hly of photobacterium mermaides_chThe protein has good immunogenicity, can be used for establishing an ELISA diagnosis method, and lays a foundation for the rapid diagnosis of the high-toxicity mermaid photobacillosis.

(2) The invention disclosesHly of photobacterium hominis_chAfter the protein is used for immunizing mice, 65 percent of the immunized mice can be protected, namely the Hly of the photobacterium mermairei in the invention_chThe protein has wide prospect in the research of mermaid photobacterium vaccine.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a diagram showing the hemolytic activity of a single colony of Photobacterium mermairei MCCC 1K03226 provided by the present invention on a sheep blood plate;

FIG. 2 is the hemolytic activity of single colony of Photobacterium mermaid MCCC 1K03226 provided by the present invention on rabbit blood plate;

FIG. 3 is a diagram showing PCR agarose gel electrophoresis for detecting hemolysin gene according to the present invention;

wherein, Lane M is Marker; lane 1 is hly_ch(ii) a Lane 2 is hly_pl(ii) a Lane 3 is dly;

FIG. 4 is a schematic representation of hly provided by the present invention_chGene PCR agarose gel electrophoresis picture;

wherein, Lane M is Marker; lane 1 is hly_ch；

FIG. 5 is a schematic diagram of Hly according to the present invention_chProtein secondary structure prediction maps;

FIG. 6 is a drawing showing hemolysin Hly according to the present invention_chSDS-PAGE electrophoretogram of the protein;

wherein, Lane 1 is the hemolysin protein Hly_ch(ii) a Lane 2 is an empty control;

FIG. 7 is a drawing showing hemolysin Hly according to the present invention_chWestern blot electrophoresis picture of protein;

wherein, Lane 1 is the hemolysin protein Hly_ch(ii) a Lane 2 is an empty control;

FIG. 8 is a drawing showing the purification of hemolysin Hly according to the present invention_chSDS-PAGE electrophoretogram of the protein;

wherein, Lane M is Marker; lane 1 shows hemolysin Hly before purification_chA protein; lane 2 shows purified hemolysin Hly_chA protein;

FIG. 9 is a Western blot analysis electrophoresis chart of serum provided by the present invention;

wherein, Lane M is Marker; lane 1 is positive serum; lane 2 is a negative control.

FIG. 10 is a drawing showing Hly according to the present invention_chMouse immunoprotection rate of protein.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Observation of hemolytic Activity of bacteria

Preparing sheep blood and rabbit blood plates respectively, picking single colony of mermaid photobacterium MCCC 1K03226, streaking on the sheep blood and rabbit blood plates, culturing at 30 ℃ for 24h, and observing hemolytic activity, wherein the results are shown in figures 1 and 2, wherein figure 1 shows sheep blood, and figure 2 shows rabbit blood. As can be seen from FIGS. 1 and 2, the bacterium was β -hemolytic on both sheep blood and rabbit blood plates, indicating that the bacterium has very strong hemolytic activity.

Example 2 recombinant vector pMD18T-hly_chConstruction of

Photobacterium damselae subsp.damselae CIP 102761(PhddCIP 102761, Gene accession number: NZ _ ADBS00000000) genome-wide sequences were downloaded from NCBI to find dly, hlyA_plAnd hlyA_chGene sequence, designing primer, the primer sequence is shown in table 1; wherein dly gene sequence is SEQ ID NO: 1; hlyA_plThe gene sequence is SEQ ID NO: 2; hlyA_chThe gene sequence is SEQ ID NO: 3. extracting the genome DNA of the strain MCCC 1K03226DNA by using a bacterial genome extraction kit, amplifying by using primers in Table 1 by using the genome DNA as a template by using touchdown PCR (polymerase chain reaction), and carrying out electrophoresis by using 1% agarose gel after the reaction is finished, wherein the result is shown in a figure 3, wherein the primers Dly-f and Dly-r are used for detecting the length of a Dly gene part and are used for detecting hemolysin Dly protein; primer Hly_pl-f test, Hly_pl-r detection of amplified Hly_plA gene portion length for detecting plasmid-encoded hemolysin; primer Hly_ch-f test, Hly_ch-r detection of amplified Hly_chGene part length for detection of chromatin-encoded hemolysin.

Table 1: primer sequence Listing

As can be seen from FIG. 1, three hemolysin Dly proteins, Hly_plProtein and Hly_chIn proteins, hly_chThe gene is detected to be positive by about 420bp, so the bacterium MCCC 1K03226DNA carries hly_chGenes without carrying hly_plAnd dly gene.

Further using the genome DNA of the strain MCCC 1K03226DNA as a template according to hly published by NCBI_chGene sequence, signal peptide prediction by Signa IP, and primer Hly_ch-F、Hly_ch-R amplifying hly from which the signal peptide coding sequence has been removed by touchdown PCR_chThe gene was full length, and the results of electrophoresis on a 1% agarose gel after completion of the reaction are shown in FIG. 4. As can be seen from FIG. 4, the amplified band size was as expected, approximately 1812 bp.

Will hly_chThe full length of the gene is connected with a T vector by a conventional method to obtain a recombinant vector pMD18T-hly_chAnd the recombinant vector pMD18T-hly_chSequencing, homology comparison is carried out on the sequencing result by using the Blastn working region of NCBI, and the amplified hly is found_chThe homology of the gene and the mermaid photobacterium hemolysin hlyA gene reaches 99 percent, so the mermaid photobacterium MCCC 1K032 is successfully obtained26 hly_chThe full length of the gene.

Further using online software ProtParam (http:// www.expasy.org/cgi-bin/ProtParam) to analyze the basic physicochemical properties; the protein subcellular localization analysis is carried out by applying online software PSORTb (http:// www.psort.org/PSORTb /), and the size of the protein coded by the gene (signal peptide is removed) is known to be 64.7 kDa; the half-life period is 20h (in vitro mammalian cells), the stability coefficient is 40.17, and the protein belongs to easily degradable protein; protein sublocalization analysis showed Hly_chThe protein is extracellular and is adapted to the biological function it performs, i.e. the protein is secreted out of the bacteria, thus causing hemolysis of the host red blood cells. Further analysis of Hly Using DNAstar_chProtein secondary structure, results are shown in figure 5. As can be seen from FIG. 5, Hly_chThe protein secondary structure comprises 14 alpha helices, 22 beta folds and 50 turns.

Example 3 recombinant expression vector pET32a-hly_chConstruction and prokaryotic expression of

The restriction enzymes BamH I and Sal I were used to treat pMD18T-hly_chAnd an expression vector pET32a (+) are subjected to double enzyme digestion, enzyme digestion products are recovered by gel electrophoresis and are mixed according to the proportion of 3:1, the mixture is connected for 2 hours at the temperature of 22 ℃, the conjugate is transformed into escherichia coli competent DH5 alpha, the escherichia coli competent DH5 alpha is coated on an LB plate containing Amp, and the mixture is cultured overnight at the temperature of 37 ℃. Picking single colony shake bacteria, and carrying out PCR identification by using a carrier universal primer. Selecting positive colony, enlarging culture, preserving strain and extracting plasmid, marking recombinant plasmid as pET32a-hly_ch。

The extracted recombinant plasmid is transferred into an expression strain BL21 to be coated on an LB solid plate containing Amp and cultured overnight at 37 ℃. Picking single colony on Amp plate, shaking bacteria overnight, sucking 1mL bacterial liquid the next day, inoculating to 100mL liquid LB culture medium containing Amp, shaking bacteria at 220r/min to OD₆₀₀When the value reaches 0.6, IPTG is added to induce expression for 4 h.

Example 4 mermaid Photobacterium hemolysin Hly_chMethod for identifying protein

And (3) centrifugally collecting the thalli, washing the thalli for 3 times by using PBS, then resuspending the PBS, adding a Loading Buffer, boiling and cracking the washed thalli, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to obtain a separation gel with the concentration of 12%, wherein after electrophoresis, carrying out gel staining, decoloring and observing results, and obtaining a result as shown in FIG. 6, wherein the target protein conforms to the expected protein size of about 79kDa, and the protein expressed by an Empty plasmid control (Empty vector) is about 20 kDa.

Meanwhile, the expression of the target protein was verified by Western blot using the His-tagged monoclonal antibody as the primary antibody at a concentration of 1:600-1000 and the goat anti-mouse IgG-HRP as the secondary antibody at a concentration of 1:1000-2000, and the results are shown in FIG. 7, in which FIG. 7 shows that the expected blot band appeared at 79kDa and in which Hly was obtained from FIGS. 6 and 7_chThe protein was expressed.

Example 5Hly_chPreparation of murine antiserum for protein and titer determination

Induction of Hly_chExpressing protein, ultrasonic cracking thallus, centrifuging, taking supernatant and precipitate, performing SDS-PAGE, and determining Hly_chThe protein is expressed by inclusion body; the protein was purified by QIAGEN Ni-NTA column, and the purification results are shown in FIG. 8, which shows that the protein purification effect is good in FIG. 8.

Adding Freund's complete adjuvant into the purified protein at a ratio of 1:1, emulsifying, and immunizing BALB/c mouse of 6 weeks old with an immunizing dose of 100 μ g/mouse; after 3 weeks, the purified protein is added with Freund's incomplete adjuvant at a ratio of 1:1 for emulsification, secondary immunization is carried out, and then, the immunization is strengthened after two weeks, wherein the steps are consistent with the secondary immunization. Wherein the dose of the secondary immunization and the booster immunization is 50 mug/mouse, blood is taken from the eyeball of the mouse after the immunization is finished, serum is separated, the serum is diluted by 1:200, and then Western blot analysis is carried out, and the result is shown in figure 9. As can be seen in FIG. 9, a clear blot band appeared at about 79kDa, consistent with the expected protein size, indicating Hly_chThe protein has certain immunogenicity.

The mermaid photobacterium MCCC 1K03226 cultured to logarithmic phase is washed and resuspended by 1mL of PBS, and Western blot analysis is performed after Loading Buffer is added for sample boiling.

And (3) coating the ELISA plate with 10 ng/hole protein, taking mouse immune serum as a primary antibody, taking an HRP-goat anti-rabbit secondary antibody as an enzyme-labeled antibody, performing ELISA (enzyme-linked immuno sorbent assay) determination, and determining the antibody titer. Through ELISA detection, the antibody titer is 1:5000, which shows that Hly_chThe protein has high immunogenicity.

Example 6Hly_chImmunoprotection evaluation of proteins

The immunization method was the same as in example 5: adding Freund's complete adjuvant into the purified protein at a ratio of 1:1, emulsifying, and immunizing BALB/c mouse of 6 weeks old with an immunizing dose of 100 μ g/mouse; after 3 weeks, the purified protein is added with Freund's incomplete adjuvant at a ratio of 1:1 for emulsification, secondary immunization is carried out, and then boosting immunization is carried out after two weeks, wherein the steps are consistent with the secondary immunization, and the doses of the secondary immunization and the boosting immunization are both 50 mu g/mouse. Two weeks after the boosting, the bacteria are detoxified, and Balb/c mouse mermaid photobacterium MCCC 1K03226 is injected into the abdominal cavity with the dosage of 5LD₅₀(i.e., 6X 10)⁷cfu), the number of dead mice was counted within one week after infection. As can be seen from FIG. 10, Hly_chAfter the protein is used for immunizing a mouse, the immune protection is high and can reach 65%.

In conclusion, the present invention relates to Hly of Photobacterium mermaides_chThe protein has good immunogenicity and immune protection, can be used for developing genetic engineering subunit vaccine, and lays a foundation for prevention and control of mermaid photobacillosis.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> Hubei institute of science and technology

<120> mermaid photorhalysin Hlych protein with immune protection effect

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1722

<212> DNA

<213> Artificial Sequence

<400> 1

atgaaaataa aaacgcttac tatgttaatt gtgtgctgct cctcaaatgc atatgctttt 60

acacaatggg gaggtagcgg tttaactcct atgggacatg aatggttaac gcgagcctca 120

gctttagagc ttcttaattc tgaggatcta ataaagccag acccagaaga tcctagactt 180

aactggacac aaggccacgc aaaaaattta gatcttgaaa tagcatcaaa tgaagtatca 240

aaaataaaga atctaaaaaa aagtgataag ctatacgaac ctaaatatga tgctattttt 300

tctgcaattg ttggtgaacg atgggtcgat attgctggtt ttaatgtaat agatggcttt 360

atcggtcaat atggcccaga ttgttttgat gcgacagcac aagaaccagc agatctccag 420

caagatcatt tcatgcgacg ttatgatgat atcggaagcc aaggaggcgt ttctgcagca 480

aaaagagctc aaaatcgatt tattgatcac tttgttaatg ctgcaatggc aaatgaaaaa 540

agaatattag tatgggatgg tggagcatca tcatcaacac aatctgaagt agactataat 600

tacttcttat ttggacgagc ggtccatttg tttcaagatt catttagcct agagcatact 660

gttcgattac cagaagataa ttataaaaaa attagacaag tcaaaggata tatttgttct 720

gacagttcag aacagcattc tcatagcagt tttacttctt ctgatttcta tgactctaat 780

gatgtaatat ggaagaaaaa atcaaaatct gataccggat ggcaatcata taaaccaagt 840

aatatgaagc cagccgcttt aactgcacta gaagcaagta aagatctatg ggctgctttt 900

ataagaacaa tggcaacaaa tattaatgaa cgggaaacta cagctagaaa agaggcacaa 960

caattaattg atacatggct atcttttgat gaggaagaaa tgttgtcatg gtatgacaac 1020

gaaaaaaatc gagatgatac atttgtaaca tctgataata aaaaaggaca atctcaaaaa 1080

acttgcttat caaacataag ctttaaaaat tcagatggtt caaagcctac acctactaac 1140

atagaagaat tagcatctaa tatagaaaag agtagaaata aatgtttatt taatattgaa 1200

cctgttcctg gtttcgctga tctatatgac ccttatatta aaatccctta taattggcag 1260

tggaaatcaa attcttggaa aacgcctggt caagattggg ctccatcaac cccaaaacct 1320

gataatggtg aagtcataaa tatattagcg tatgacaata atcagctttc agcagatact 1380

attgcaaata attcgaaaat tataacatca ggtaagaaag gtctagattt cataaaagta 1440

ccatcagaga ataatggtta ttatttcaga ctaaataatt accctaattt attctttagc 1500

tattcagcta gtgcagatgg aactgttaaa ttagtaaact cacctaaaca atcagagttt 1560

attttaatag gaaataaaaa cacatataac ataaagaaca catattggga tcagttcgta 1620

tggtataata aagctaaaaa atcagtacat ttaacgtctc atggtaacga aaaaaacaca 1680

gactcgtcat ggacaatatt aaataaagat attaataact ag 1722

<210> 2

<211> 1812

<212> DNA

<213> Artificial Sequence

<400> 2

atgaaaatta gaaaactata tagttgtata cttttaggtc ttagtagcct atccgctagt 60

gctatagccg aagttgataa ctattctaca cctgcagacg tagtgtcaat actaagcagt 120

gtaaaaaatc cagatcgtat tgtatatata aatatgaaac aggaggaact atcaacctac 180

gattctatat tagaagatat cataaataat gacaaacaat acatctttga tctttctttt 240

gatagtgatg aagaaaaagc taatcttcaa aagaaattta aggatttaat gggagtaaaa 300

tttgatagta attttatagt tgtaactggc tataaaaatc aactaatgta tactccgata 360

tcagatacta atgatcgtat gatatctatt ctagatcatg aagctaaatc taatgatata 420

tctaaattcc ctagagcttt agccttaagt tcaggtaaag ttgctagtga tcaatcaatt 480

tctttgcccc atgtttcgtt ttaccttaat gtcaataaag aaataactga tcaggaatgt 540

actttccgaa attctttaat ttgggataga ggaagtcgtt ctttctgtaa aaatggtaac 600

atttcactta tttatcaagt tatattagag cgttctttat catttggtac taatggcgtt 660

gcaactcctg atgcaaaaat agttcgaatt agcttagatg ataacactac tggtgccggt 720

atacatctaa atgatacact gacacataaa tcctattggg ctaattatca agtggtatct 780

ggttgggcta gagaatggtc agccagtgct attgctcaag attatttatt tgatatatcc 840

acttcaaatg aaaaagctca gatattaaaa acgttccctc gtgagaatat taattctaat 900

tataatataa atgaatcttc tggattcacc attggcgtta ctggtggtgc agaggtgagt 960

aaagatggac cgaaagctag tcttcaagct agtgctagtt atagtcaatc aaaatcatta 1020

tcttttaata cacaagatta tagagttgaa aagaattcaa cttctgcaca aaatgtttct 1080

ttccgttggg ctagagaaca atatcctaac tcagagtcat tgttagataa gtggacaaat 1140

cctgtatggt cagaagatta tcctgcaaat ctgaaaaaag ttcagcctct tagttatgcg 1200

agttttgttc ctaaacttga tgtcatatat aaagcttctc ctaatgaaac aggaaaaact 1260

caatttacta tagattcatc agttaatatt atgcctctat ataatagagc atggttctat 1320

ttttatggag ttggcgcaca ccagacctat tatggagtag atgaccaacc gcttcgtaga 1380

gttaacaaag caatatcatt tactgttgat tgggagcacc ctgtgtttac tggaggaacc 1440

ccagtaaata tacagttggc ttcttttaac aataaatgta tggatataca aaataaaggg 1500

atagtaatga ccgctgaatg tgatattaat agcaaatcgc agtcatttat ttatgatcaa 1560

tataatcgat acgttagtgc aactaatact aaactatgtt tggatggtga atctctatcg 1620

gaattacaac cttgtagttt gaagctaact caaaaatggt tatgggatgg agataaatta 1680

aaaaatagtt ttgaagataa atacttaact tttgataatg atactttctt attagaatct 1740

agcttatcta ataaacaaaa attaaattca tcatatacaa gagtttttga ttcatcgact 1800

ataaataagt aa 1812

<210> 3

<211> 1812

<212> DNA

<213> Artificial Sequence

<400> 3

atgaaaatta gaaaactata tagttgtata cttttaggtc ttagtagctt atctgccagc 60

gctatagctg atgttgataa ttattctacg ggcgcagacg ttatatcaat attaagcagt 120

gtgcaaaatc cagatcgtat tgtatatgta aatatgaagc aggaggaaat atcaacttac 180

aaccgtatat tagaagatat catatataat gataaacaat atatctttga tctttctttt 240

gataataatg aagaaaaaga gaagcttcaa aagaaattta aggatttaat gggagtaaaa 300

tttgatagta attttatagt tgtaacaggc tataaaaatc aactaatgta tactcctata 360

gcagatgcta atgatcgtat ggtatctgtt ctagatcatg aagccagatc taatgatatc 420

tctaaatacc caatggcttt agctttacgt tcaggtaacg ctactagtga tcaatcaatt 480

tctttgcccc atgtttcgtt ttatcttaat gttaataaag aaataactga tcaggaatgt 540

actttccgaa attctttaat ttgggataga ggaagtcgtt ctttctgtaa aaatggtaat 600

atttcactta tttatcaagt tatattagag cgttctttat cgtttggtac taatggcatt 660

gcaacgcctg atgcaaaaat agttcgaatt agcttagatg ataatacgac aggtgccggt 720

atacatctaa atgatacact gacgcaaaaa tattattggg ctaattatca ggtggtatct 780

ggttgggcta gagagtggtc agccagtgct attgctcaag attatttatt tgatatatct 840

acttcaaata aaaaagctca gatattaaaa acgttccctc gggagaatat taattctaat 900

tataatataa atgaatcttc tggattcacc attggtgtta ctggtggtgc tgaagttagt 960

aaagatggcc cgaaagcgag tcttcaggct aatgctagtt atagtcaatc taagtcgtta 1020

tcttttaata cccaagacta tagagttgaa aaaaattcaa cttctgccca aaatgtttct 1080

ttccgttggg cgagagagca gtatcctgac tcagagtcac tgttagataa gtggacaaac 1140

cctgtatggt cggaaggtta tcctgcgaat ctaaaaaaag ttcagccgct tagttatgcg 1200

agttttgttc ctaaacttga tgttatatac aaagcatctc ctaatgaaac aggaaaaact 1260

caatttacta tagattcatc agttaacatt atgcctctat ataatagatc gtggttctat 1320

ttttatggaa ttggtgcaca tcagacttat tatggtgtag atgatcagcc tcttcgtaga 1380

gttaataagg caatatcatt tactgttgat tgggagcacc ctgtatttac tggtggaact 1440

ccggtaaatt tacagttagc atcttttaat aataaatgta tcgatatcca aaacaacaat 1500

aaagtaatga cggctgaatg tgatattaat agtaaatcac agtcatttat ttatgaccaa 1560

tataatcgat atgttagcgc aactaatact aaattatgct tagatggcga atctttatca 1620

gaactgcaat cttgtagctt gaagctaacg caaaaatggt tatgggatgg agatagatta 1680

aaaaatagtt ttgaagataa atacttaact gttgatagtg atactttatc gttagaaact 1740

ggtttatcta ataaacaaaa attaaattca tcatatacaa gagtttttga cccatcaacc 1800

ataaatcagt aa 1812

<210> 4

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 4

taactcctat gggacatgaa tgg 23

<210> 5

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 5

tatagatcag cgaaaccagg aac 23

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 6

agcctcttag ttatgcgagt t 21

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 7

cggtcattac tatcccttta tt 22

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 8

atttactgtt gattgggagc 20

<210> 9

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 9

gatttatggt tgatgggt 18

<210> 10

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 10

gcggatccga cgttatatca atattaagca g 31

<210> 11

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 11

gcgtcgactt actgatttat ggttgatggg tc 32

Claims (5)

1. Mermaid photorhabditis hemolysin Hly with immune protection effect_chThe application of the protein in the development of the mermaid photobacterium genetic engineering subunit vaccine is characterized in that the preparation method of the subunit vaccine comprises the following steps:

1) recombinant vector pMD18T-hly _ch Construction of

Extracting genome DNA of the photobacterium mermairei, wherein the strain preservation number of the photobacterium mermairei is MCCC 1K03226, the genome DNA is used as a template, and Hly is used_ch-F，Hly_ch-R is primer amplification for removing signal peptide coding sequencehly _ch A gene ofhly _ch The gene sequence is SEQ ID NO: 3; amplification Using touchdown PCRhly _ch Full length of gene, the primer Hly_ch-F、Hly_ch-the sequence of R is as follows:

Hly_ch-F：5’-GCGGATCCGACGTTATATCAATATTAAGCAG-3'; has BamHI enzyme cutting site; SEQ ID NO: 10;

Hly_ch-R：5’-GCGTCGACTTACTGATTTATGGTTGATGGGTC-S'; carrying SalI enzyme cutting site; SEQ ID NO: 11;

the said obtainedhly _ch The gene is connected with a T vector to obtain a recombinant vector pMD18T-hly _ch And the recombinant vector pMD18T-hly _ch Sequencing and protein subcellular localization;

2) recombinant expression vector pET32a-hly _ch Construction of

The restriction enzymes BamH I and Sal I were used for the restriction enzyme BamH I and Sal I, respectivelyThe recombinant vector pMD18T-hly _ch Carrying out double enzyme digestion with an expression vector pET32a (+), recovering the enzyme digestion product by gel electrophoresis, mixing the enzyme digestion product according to the proportion, connecting the enzyme digestion product for 2 to 3 hours at the temperature of between 22 and 25 ℃, transforming escherichia coli competent cells, inoculating the escherichia coli competent cells into an LB plate I, and culturing the escherichia coli competent cells overnight at the temperature of 37 ℃; picking single colony shake bacteria, and performing PCR identification by using a carrier universal primer; the positive colony is expanded and cultured, the strain is preserved, and the plasmid pET32a is extractedhly _ch ；

3）hly _ch Prokaryotic expression of genes

The plasmid pET32a-hly _ch The transformed expression strain BL21 is spread on a second LB plate and cultured overnight at 37 ℃; picking the single colony of the second LB plate and shaking the bacteria overnight, inoculating the bacteria liquid into the liquid LB culture medium the next day, shaking the bacteria at 220r/min to OD₆₀₀Adding IPTG to induce expression for 4-6 hr when the value reaches 0.6-0.8;

4）Hly_chpreparation of protein subunit vaccine

Induction of Hly_chAfter protein expression, carrying out ultrasonic lysis on thalli, centrifuging, and purifying protein by using a QIAGEN Ni-NTA column to obtain target protein; mixing Hly_chEmulsifying the protein with Freund's complete adjuvant to obtain subunit vaccine;

wherein the competent cells are DH5 alpha, the first LB plate and the second LB plate are solid LB plates containing ampicillin, and the concentration of the ampicillin is 45-55 mu g/L;

the liquid LB in the step 3) is liquid LB containing ampicillin, the concentration of the ampicillin is 45-55 mu g/L, and the concentration of IPTG is 1 mM.

2. The immunoprotective photorhabdus mermairei hemolysin Hly of claim 1_chThe application of the protein in the development of the mermaid photobacterium gene engineering subunit vaccine is characterized in that the recombinant vector pMD18T-hly _ch The ratio of the product recovered by the restriction enzyme digestion of the expression vector pET32a (+) is 3: 1.

3. the immunoprotective photorhabdus mermairei hemolysin Hly of claim 1_chThe protein is applied to development of mermaid photobacterium genetic engineering subunit vaccines, and is characterized by also comprising SDS-PAGE analysis and Western blot verification;

centrifuging the bacterial liquid in the step 3) of the claim 1, collecting thalli, washing the thalli for 3 times by PBS, then suspending the thalli, adding a Loading Buffer to boil and crack the thalli, carrying out SDS-PAGE electrophoresis, and after the electrophoresis is finished, carrying out gel dyeing and decoloring for observing results; and simultaneously carrying out Western blot to verify the expression of the target protein.

4. The immunoprotective photorhabdus mermairei hemolysin Hly of claim 3_chThe protein is applied to development of mermaid photobacterium genetic engineering subunit vaccine, and is characterized in that the concentration of separation gel in SDS-PAGE electrophoresis is 12%.

5. The immunoprotective photorhabdus mermairei hemolysin Hly of claim 3_chThe protein is applied to development of a mermaid photobacterium genetic engineering subunit vaccine, and is characterized in that in the Western blot method, the primary antibody is a monoclonal antibody with a His label, and the secondary antibody is goat anti-mouse IgG-HRP.

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