CN110628796B

CN110628796B - Fish Nocardia disease common antigen DNA vaccine and preparation and application thereof

Info

Publication number: CN110628796B
Application number: CN201910418664.2A
Authority: CN
Inventors: 鲁义善; 夏立群; 陈建林; 王文基; 李贝
Original assignee: Shenzhen Yihai Biotechnology Co ltd; Guangdong Ocean University; Shenzhen Research Institute of Guangdong Ocean University
Current assignee: Shenzhen Yihai Biotechnology Co ltd; Guangdong Ocean University; Shenzhen Research Institute of Guangdong Ocean University
Priority date: 2019-05-20
Filing date: 2019-05-20
Publication date: 2023-03-24
Anticipated expiration: 2039-05-20
Also published as: CN110628796A

Abstract

The invention provides a fish Nocardia disease common antigen DNA vaccine and preparation and application thereof, wherein the preparation method comprises the following steps: comparing the immunoblotting results of the three fish Nocardia by using a two-dimensional electrophoresis technology, and screening common immunoblotting protein points according to the isoelectric points and the molecular weights; according to the isoelectric point and the molecular weight of the common antigen, cutting off the corresponding protein point for mass spectrometry and N-terminal sequencing; identifying coding genes of the common antigen by combining mass spectrometry and protein sequencing; selecting common antigen candidate genes after mutual verification according to the common antigen encoding genes and encoding genes of vaccine candidate common antigens predicted by reverse vaccinology; designing a primer according to the sequence of the common antigen candidate gene, and cloning the primer into a vector to obtain the eukaryotic expression recombinant plasmid. The DNA vaccine prepared by the invention has prevention and treatment effects on fish nocardiosis in different degrees.

Description

Fish Nocardia disease common antigen DNA vaccine and preparation and application thereof

Technical Field

The invention relates to the field of vaccines, in particular to a fish Nocardia disease common antigen DNA vaccine and preparation and application thereof.

Background

The nocardiosis of fishes is one of common bacterial infection diseases in pond culture, raft type net cage culture and deep water net cage culture modes, belongs to a chronic, systemic and granulomatous disease, and is commonly called as sarcoidosis. The nocardiosis pathogenic bacteria isolated in fish so far include three types: nocardia seriolae (n.seriolae), nocardia stardana (n.asteroids), and nocardia salmonicida (n.salmonicida). The nocardia of the fishes is widely distributed in soil, oceans, rivers, activated sludge, animal excrement and rotten vegetation, and can be used for infecting fishes with low immunity and weak constitution through baits, gills or wounds and the like to induce bleeding and ulceration of the body surfaces of the fishes and full spreading of a large number of white nodules on internal organs. According to research and analysis of some fishes, seriolala infected with nocardiosis shows body surface bleeding, lip erosion, granulomatous bulge on abdomen and white nodule on liver, spleen, kidney and heart; some diseased fishes, such as seriola quinqueradiata, have concave-convex surface, exuviation bleeding and abscess, and have a large number of large nodules in internal organs (such as kidney, spleen and liver), wherein some diseased fishes only have small nodule diseases in gill parts; some diseased fishes, such as scad, have small nodules and hemispherical bulges on the body surface, and numerous castellated nodules spread over the spleen and kidney, and some have pyogenic infections between the lumbar vertebral bodies of some diseased fishes. The pathogenic bacteria of nocardia (N.seriolae) of fish have a long infection period, and early infection is usually in recessive symptoms and is extremely difficult to find; the typical symptoms and hazards typically occur during the adult fish stage. The diseased fish generally shows that the body surface is easy to rub and bleed, the reaction speed is slow, the swimming equilibrium is poor, and white nodules, nodular pustules and other symptoms appear on the body surface and internal organs, and finally the death phenomenon appears. Nocardia is widely prevalent in the global aquaculture industry, can infect a variety of freshwater and marine fishes, and is a snakehead (Channa argus) cultivated in korea; culturing Seriola quinqueradiata (Seriola quinqueliana) in Japan; oncorhynchus major (Oncorhynchus tsawawytscha) cultured in Europe and America; one of the most major bacterial infection pathogens, such as channa maculata (c. Macular) bred in taiwan area, scortum barcoo (Terapon jarbaua), micropterus salmoides (Micropterus salmoides) bred in mainland area, pachinko (ospermus goramy), trachinotus ovatus (Trachinotus ovatus), large yellow croaker (larvicea), channa argus (c.argus) and channa maculata (c.macular) bred in mainland area. Nocardiosis of fishes has become stumbling stone on various fish industrialized culture roads, which causes increasingly serious economic loss of the aquaculture industry all over the world.

Traditionally, antibiotics with sensitivity to pathogenic bacteria are generally adopted in aquaculture to treat and prevent bacterial infection diseases of fishes. The nocardia of the fish belongs to facultative intracellular parasitic bacteria, can be phagocytized by macrophages but not killed after infecting the fish body, is hidden in nodules of the diseased fish body, and can not be effectively killed by the drugs entering the nodules; and the abuse problem of antibiotics is increasingly serious, so that a series of irreversible influences are caused, such as environmental pollution, water body degradation, probiotic flora imbalance, fish body drug residue and the like. Therefore, in order to avoid the problems and effectively control the occurrence and prevalence of fish nocardiosis, the development of high-efficiency and environment-friendly vaccines is not easy. To date, the development of vaccines against nocardiosis in fish is not ideal. Related researches show that various inactivated vaccines of the nocardia seriolae can improve the titer of serum agglutination antibodies, but basically cannot provide any immune protection effect after challenge, so that the feasibility of the development route of the nocardia seriolae inactivated vaccine is lost. The mechanism that the nocardia seriolae inactivated vaccine has no immune protection is not clear, and probably because nocardia seriolae has an immune escape mechanism similar to antigen variation. Itano et al, using several non-pathogenic bacteria of Nocardia as live vaccines, can produce a certain protection rate against Nocardia seriolae, but the relative protection rate is only 65.4% at most, and have no practical application value, probably because the natural antigens of Nocardia pathogenic bacteria and related non-toxic species of the Nocardia pathogenic bacteria are different greatly. In general, the current vaccine research on fish nocardiosis is in a predicament. Therefore, the preparation of novel vaccines with high efficiency and safety by using modern molecular biology technology is very necessary, which represents the leading edge and trend of the development of international aquaculture industry at present.

Disclosure of Invention

The invention provides a common antigen DNA vaccine of three pathogenic bacteria of fish nocardiosis, which has obvious immune protection effect on fish and can effectively prevent the fish nocardiosis from invading. The vaccine is simple and convenient to operate, safe to use, remarkable in effect and has practical commodity development and application values.

The invention provides a preparation method of a DNA vaccine for fish nocardiosis, which comprises the following steps: performing whole-bacterium protein electrophoresis on Nocardia seriolae, nocardia asteroides and Nocardia salmonicida respectively by using a two-dimensional electrophoresis technology, comparing the immunoblotting results of the three kinds of Nocardia seriolae, screening common immunoblotting protein points according to isoelectric points and molecular weights, wherein the common immunoblotting protein points are common antigens; performing two-dimensional electrophoresis of the three nocardia whole proteins respectively, and cutting corresponding protein points on electrophoresis gel after dyeing by Coomassie brilliant blue according to the isoelectric points and molecular weights of common antigens to perform mass spectrometry and N-terminal sequencing; identifying coding genes of the common antigen by combining mass spectrometry and protein sequencing; selecting a common antigen candidate gene after mutual verification according to the common antigen coding gene and a coding gene of a vaccine candidate common antigen predicted by reverse vaccinology; designing a primer according to the sequence of the common antigen candidate gene, and cloning the primer into a vector to obtain the eukaryotic expression recombinant plasmid.

In the above preparation method, further comprising: culturing the engineering strain containing eukaryotic expression recombinant plasmid, and largely extracting recombinant plasmid without endotoxin.

The invention also provides a DNA vaccine for the fish Nocardia disease obtained by the preparation method.

The invention also provides application of the DNA vaccine of the fish Nocardia disease in preventing and treating the fish Nocardia disease.

The common antigen DNA vaccine prepared by the invention has obvious immune protection effect on fish and can effectively prevent the fish from being invaded by nocardiosis.

Drawings

FIG. 1 shows two-dimensional electrophoresis and immunoblotting of total mycoprotein of three pathogenic bacteria of nocardiosis seriolae, wherein A, C and E are SDS-PAGE electrophoresis images of total mycoprotein of nocardia seriolae, nocardia salmonicida and nocardia asteroides respectively; b, D and F are respectively Nocardia seriolae, nocardia salmonicida and Nocardia asteroides holothurian 2D wsster blot blots.

Detailed Description

The following examples are presented to enable those skilled in the art to more fully understand the present invention and are not intended to limit the invention in any way. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all purchased from conventional biochemicals, unless otherwise specified.

The DNA vaccine is a novel vaccine after the traditional vaccine (attenuated live vaccine, inactivated vaccine) and the genetic engineering subunit vaccine, the technology directly injects exogenous gene recombination eukaryotic expression plasmids which encode certain antigen protein into an animal body, and the foreign gene recombination eukaryotic expression plasmids rely on an expression system of host cells to synthesize the antigen protein and stimulate the immune response of an organism, thereby achieving the effect of treatment. Compared with the traditional vaccine which has the safety problem that the virulence of pathogenic bacteria is back strong or the inactivation is incomplete, and the defects of low immunogenicity and high production cost of subunit vaccine, the DNA vaccine has the unique advantages of low production cost, capability of forming multivalent vaccine with other immunogen, capability of inducing organism to generate strong and durable humoral immunity and cellular immunity, good safety and the like, and simultaneously, the inoculation way is diversified, and the DNA vaccine can adopt an injection way for inoculation, and can also be orally taken, sprayed and the like. In addition, the DNA vaccine has the advantages of low immunization dose, long duration and the like, and long-acting immune protection can be obtained by inoculating an organism once without repeatedly strengthening immunity for many times. Therefore, the development of the Nocardia seriolae DNA vaccine has important significance for preventing and treating the Nocardia disease of the fishes.

The invention aims to screen a common antigen of all mycoprotein of three pathogenic bacteria of nocardiosis seriolae (nocardia seriolae, nocardia asteroides and nocardia salmonicida) and prepare a DNA vaccine.

The technical scheme adopted by the invention is as follows:

1. screening and identification of common antigens of Nocardia seriolae, nocardia asteroides and Nocardia salmonicida

a. Performing whole-bacterium protein electrophoresis on Nocardia seriolae, nocardia asteroides and Nocardia salmonicida respectively by using a two-dimensional electrophoresis technology, performing Western blot by using Nocardia seriolae specific antibodies as primary antibodies after the electrophoresis gel is subjected to membrane conversion; comparing the immunoblotting results of 3 kinds of fish Nocardia, and screening the common immunoblotting protein spots (common antigens) according to the isoelectric points and the molecular weights.

b. Respectively carrying out full-bacterial protein two-dimensional electrophoresis of 3 Nocardia under the same conditions, and cutting corresponding protein points on electrophoresis gel according to the isoelectric points and molecular weights of common antigens after Coomassie brilliant blue staining to carry out mass spectrum analysis and N-terminal sequencing.

c. Coding genes of common antigens are identified by combining mass spectrometry and protein sequencing according to an access number NZ _ JNCT01000022 of Nocardia seriolae whole genome sequencing database.

2. Selection, cloning and expression of candidate genes for nucleic acid vaccines

a. And (3) predicting and screening the candidate common antigens of the vaccine by using a reverse vaccine analysis method according to the whole genome sequence information of the nocardia seriolae.

b. And selecting the common antigen candidate gene after mutual verification according to the common antigen encoding gene and the encoding gene of the vaccine candidate common antigen predicted by reverse vaccinology. Primers are designed according to the sequences of the candidate genes and cloned into pcDNA 3.1-FLAG vector to obtain eukaryotic expression recombinant plasmid.

3. Development of nucleic acid vaccine and evaluation of immunogenicity

a. Culturing engineering strain containing eukaryotic expression recombinant plasmid, extracting recombinant plasmid without endotoxin in large amount, preparing common antigen DNA vaccine, and immunizing hybrid snakehead by intramuscular injection.

b. After each group of DNA vaccines are immunized with hybridized snakehead for 35 days, a nocardia seriolae wild strain is inoculated in an intraperitoneal injection mode, 14 days are continuously observed, the death mantissa is counted, and the immune protection rate is calculated.

The following description of specific embodiments is provided to enable those skilled in the art to better understand the invention.

Experimental Material

(I) Strain

Wild strain of Nocardia seriolae (N.seriolae strain ZJ 0503) is separated from diseased trachinotus ovatus (T.ovatus) cultured in Yangjiang net cages and stored in key laboratories of pathogenic biology and epidemiology of economic animals of Guangdong province. A related article is published in L.Xia, J.Cai, B.Wang, et al.draft Genome sequence of Nocardia seriolae ZJ0503, a fish pathology isolated from achieves ovatus in China [ J ]. Genome intersections, 3 (2015).

Nocardia asteroids is purchased from China center for culture Collection of microorganisms, and the strain number is ATCC 19247.

The Salmonella choleraesuis N.salmonicida is purchased from China center for culture Collection of microorganisms with the culture deposit number being ATCC 27463.

(II) reagent

(1) Improving a culture medium:

sterilizing at high temperature and high pressure, and adding 2% agar to obtain modified culture medium.

(2) Lysis buffer (Lysis buffer):

storage at-20 ℃ (dithiothreitol, IPG buffer, protease inhibitor and ribozyme inhibitor are currently used)

(3) Washing (Wash buffer):

(4) 4 × separation gel buffer (4 × Tris-HCl):

(5) 10% SDS solution:

sodium dodecyl sulfate SDS 15g

ddH ₂ O 150mL

For preparing 10 Xelectrophoresis buffer and SDS equilibrium buffer

(6) 10 × electrophoresis buffer:

storing at room temperature (diluting to 1 × electrophoresis buffer for preparing agarose sealing liquid)

(7) SDS equilibration buffer:

storing at the temperature of minus 20 ℃, and subpackaging into 10 mL/tube, and 20 tubes in total;

1% DDT and 2.5% Iodoacetamide Iodoacetamide before use

(8) Agarose sealing liquid:

1 × 50mL of electrophoresis buffer

Agarose agar 0.25g

1% Bromophenol blue Bromophenol blue 100. Mu.L

Storing at normal temperature; the mixture was split into 1 mL/tube and 50 tubes in total.

(9) Hydration liquid:

IPG, DTT were added last. Finally, the mixture is split into 500 ul/tube, and 10 tubes in total.

(10) Protein fixing solution:

acetic Acid glacial Acetic Acid 100mL

Methanol 400mL

ddH ₂ O to 1000mL

(11) Coomassie brilliant blue dye liquor:

(13) A rabbit polyclonal antibody (primary antibody) against nocardia seriolala, prepared by the laboratory entrusted to shanghai friend biotechnology limited, the preparation method is a conventional method, and the method is as follows: after the Nocardia seriolae is cultured to a logarithmic phase, formaldehyde with the final concentration of 4 percent is added into the culture solution, and the Nocardia seriolae is inactivated by shaking incubation at 4 ℃. And after inactivation, carrying out 2-3 times of centrifugal collection on the thalli, washing with PBS, and adding a proper amount of PBS to prepare a bacterial suspension. New Zealand white rabbits are immunized with Nocardia seriolae bacterial suspension, and each rabbit is immunized 4-5 times at an interval of 1-2 weeks. Sera were isolated after sacrifice to obtain a rabbit polyclonal antibody (primary antibody) against nocardia seriolae. The primary antibody can be prepared by inactivating bacterial antigens.

(14) HRP-labeled goat anti-rabbit IgG (secondary antibody) Wuhan Dr bioengineering, inc.

(15) The whole mycoprotein extraction Kit 2D Clean up Kit and the whole mycoprotein purification Kit 2D Quant Kit are purchased from GE Healthcare Life Science (general electric appliances, USA) department of Life sciences of medical group.

Example 1 screening and identification of common antigens of Nocardia seriolae, nocardia asteroides and Nocardia salmonicida

Preparation of fish nocardiosis three pathogenic bacteria holomycoprotein

(1) Taking the Nocardia seriolae wild strain, nocardia asteroides and Nocardia salmonicida glycerin preservation bacterial liquid out in a refrigerator at the temperature of minus 80 ℃, putting the Nocardia seriolae wild strain, nocardia asteroides and Nocardia salmonicida glycerin preservation bacterial liquid in a constant-temperature water bath kettle at the temperature of 37 ℃ for melting, sucking 100 mu L of bacterial liquid, coating the bacterial liquid on an improved solid culture medium, putting the improved solid culture medium in a constant-temperature incubator at the temperature of 28 ℃, and culturing for 48 hours to recover the bacterial liquid; selecting single colony to prepare bacterial suspension, coating and inoculating the bacterial suspension to an improved solid culture medium, culturing in a large scale, and collecting thalli when the bacteria reach logarithmic growth phase.

(2) The three pathogenic bacteria of the wild strain of the nocardia seriolae, the nocardia asteroides and the nocardia salmonicida are gram-positive bacteria with thicker cell walls, and are respectively pretreated for 2h, 3h and 2h by using 1mg/mL lysozyme. After the treatment was completed, the cells were washed three times with a sterile PBS solution.

(3) Respectively sucking 1mL of the lysozyme-treated bacterial liquid into 1.5mL of centrifuge tubes, and centrifuging at 4 ℃ of 7000r/min for 5min; the supernatant was discarded and the precipitate was collected.

(4) Resuspending the Wash buffer three times, and centrifuging for 5min at 4 ℃ at 7000r/min each time; the supernatant was discarded and the precipitate was collected.

(5) Lysis buffer was prepared. Protease inhibitors, ribozyme inhibitors, IPG and DTT each account for 1% of the total lysine buffer system.

(6) 1mL of lysine buffer solution was added to the pellet and resuspended.

(7) The mixture is placed on ice for cracking treatment for 2h.

(8) After the lysis treatment was completed, the cells were centrifuged at 13000r/min at 4 ℃ for 1 hour, and the supernatant was aspirated and stored at-80 ℃ (the supernatant was a protein sample).

Purification of complete bacterial protein of (di) nocardia

Purifying three pathogenic bacteria whole mycoprotein of Nocardia seriolae, nocardia asteroides and Nocardia salmonicida by adopting a 2D Clean-Up kit, treating all the steps on ice, and referring to the kit specification for specific operation steps.

(III) determination of Whole bacterial protein concentration

And (3) determining the total mycoprotein concentration of the purified total mycoprotein of three pathogenic bacteria, namely Nocardia seriolae, nocardia asteroides and Nocardia salmonicida by adopting a 2D Quant kit, wherein the specific operation steps refer to the kit specification. Each group of whole eggs was divided into two equal samples to ensure that each group of experiments showed SDS-PAGE gel and Western Blot NC membrane results from the same protein sample.

(IV) one-dimensional isoelectric focusing (IEF) electrophoresis

(1) The volume of protein to be added (total protein content: 120. Mu.g) was determined from the protein concentration, and the corresponding hydration solution (1% DTT added in the hydration solution when used; hydration solution volume = 125. Mu.L-protein volume) was added to the protein solution, followed by addition of 6.25. Mu.L IPG and 1.25. Mu.L Protease inhibitor Protease Mix.

(2) And adding all mixed samples into an electrophoresis tank, covering with an adhesive tape, removing bubbles to uniformly spread the protein solution, and sucking about 1.5mL of glycerol to cover the adhesive tape.

(3) Placing the loaded adhesive tape in an Ettan IPGphor III horizontal electrophoresis apparatus for hydration for 12h at the temperature of 20 ℃; a one-dimensional isoelectric focusing (IEF) electrophoresis was then performed according to the procedure (S100V 1h, S200V 1h, S500V 1h, G1000V 1h, G10000V 3h, S10000V 8 h). After electrophoresis, the IPG strip was quickly removed and equilibrated in equilibration solution A (0.2 g DTT in 20mL SDS equilibration buffer) for 15min, and then equilibrated in equilibration solution B (0.5 g iodoacetamide in 20mL SDS equilibration buffer) for 15min.

(V) two-way polyacrylamide gel (SDS-PAGE) electrophoresis

And (3) loading the balanced adhesive tape to SDS-PAGE separation gel, sucking 100 mu L of agarose sealing liquid to cover the adhesive tape, removing air bubbles, transferring the adhesive tape to a vertical electrophoresis tank after the adhesive tape is solidified, and performing second-direction polyacrylamide gel electrophoresis according to the procedures (90V 30min, 120V 120min).

(VI) immunoblotting (Western blot) assay

And taking out the gel after the polyacrylamide gel (SDS-PAGE) electrophoresis is finished, rinsing the gel in a membrane transferring buffer solution for a plurality of seconds, opening an electric transfer clamp, filling a special sponge pad soaked by the membrane transferring solution on each side, respectively placing a rapid and efficient protein transferring pad (qualitative filter paper) soaked by the membrane transferring solution, spreading the SDS-PAGE gel on the negative side filter paper, covering the gel with a nitrocellulose NC membrane soaked by the membrane transferring solution, removing bubbles, finally sequentially adding the filter paper and the sponge, clamping and transferring the gel to a transfer tank, and transferring according to the program (150 mA and 120min). After the transfer printing is finished, placing the NC membrane in a closed liquid chamber for temperature sealing for 1h; primary antibody (1; washing with the membrane-washing solution for 10min for 4 times; adding a secondary antibody (1; washing with the membrane-washing solution for 10min for 4 times; and exposing and developing by using a DAB reagent.

(VII) Coomassie brilliant blue staining

And after the polyacrylamide gel (SDS-PAGE) electrophoresis is finished, placing the SDS-PAGE gel in a protein fixing solution, horizontally shaking for fixing for 30min, pouring out the fixing solution after the fixing is finished, and adding Coomassie brilliant blue for dyeing for more than four hours. Pouring out the Coomassie brilliant blue staining solution carefully, and changing the destaining solution for destaining until the background of the SDS-PAGE gel is clear and the protein spots are highlighted.

(VIII) immunoblot Co-protein dot screening

The SDS-PAGE gel and the Western blot NC membrane were Image-scanned by a protein gel electrophoresis Scanner Image Scanner III with an optical resolution of 300dpi. The electrophoresis patterns and transfer patterns of three pathogenic bacteria, i.e., nocardia seriolae wild strain, nocardia starchy and Nocardia salmonicida, were analyzed by ImageMaster 2D Elite 5.0.

The total proteins of three pathogenic bacteria, i.e. wild strains of Nocardia seriolae, nocardia starchy and Nocardia salmonicida are subjected to a first-way isoelectric focusing electrophoresis (IEF) and a second-way polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis test, and most of protein points are distributed between isoelectric points of 4-7 and molecular weights of 20-100kDa (shown as A, C and E in figure 1). By Western bolt test, 11 identical immunoblot sites were found on the membranes of three pathogenic holothrin Nitrocellulose (NC) respectively (shown in FIG. 1 as B, D, F). Respectively deducting 11 same immunoblotting protein spots on SDS-PAGE gels of the three pathogenic bacteria, and sending the immunoblotting protein spots to Shenzhen micronano phenanthrene biotechnology Limited for mass spectrum identification.

(nine) database analysis

Using reverse vaccinology, the NCBI BLAST (https:// BLAST. NCBI. Nlm. Nih. Gov/BLAST. Cgi) analysis was performed in combination with the Nocardia seriolae stain ZJ0503 whole genome sequence and online alignment according to mass spectrometric identification results. The result shows that 7 same antigen proteins are respectively deducted from protein samples with 11 isoelectric points and the same protein molecular weight sites, and the protein samples are respectively the No.1 point: molecular chaperone DnaK (Molecular chaperone DnaK, dnaK), point 2: molecular chaperone GroEL (GroEL), point 3: 30S ribosomal protein S1 (30S ribosomal protein S1, rpsA), point 6: terd family protein (Terd family protein, terd), point 8: FHA domain-associating protein (FHA), point 9: 50S ribosomal protein L7/L12 (50S ribosomal protein L7/L12, rplL) and point 10: pspA/IM30 family protein (PspA/IM 30 family protein, pspA).

EXAMPLE 2 construction of eukaryotic expression vectors for candidate genes

(one) primer design

According to the sequence of GroEL, rpsA, dnaK, terD, FHA, rplL and PspA genes in the Nocardia seriolae N.seriolae strain ZJ0503 whole genome (Accession number: JNCT 01000019) and the MCS enzyme cutting site of eukaryotic expression plasmid pcDNA 3.1-FLAG, primers (table 1) for constructing eukaryotic overexpression plasmids are designed by using Primer premier 5.0 software, and the primers are synthesized by the company Limited in the biological engineering (Shanghai). The sequences of the GroEL, rpsA, dnaK, terD, FHA, rplL and PspA genes are respectively shown as 29-35 sequences in a sequence table, and the corresponding protein sequences are respectively shown as 36-42 sequences in the sequence table.

TABLE 1

Note: the underlined sections are restriction enzyme sites.

Cloning, double enzyme digestion, connection and transformation of 7 common antigen candidate genes

Extracting Nocardia seriolae N.seriolae strain ZJ0503 total genome by using a bacterial genome DNA extraction kit (Tiangen) as a PCR amplification template, and performing PCR amplification by using high-fidelity polymerase. The reaction system and the amplification procedure were as follows:

the PCR annealing temperatures were GroEL:66.7 ℃, rpsa:67.5 ℃ DnaK:71.4 ℃, terD:66.85 ℃ and FHA:66.9 ℃ and RplL:63.9 ℃ and PspA:64.1 ℃. PCR products of 7 candidate genes are detected by 1% agar gel electrophoresis respectively, and the size of the target band is determined. The PCR product is purified by a DNA fragment purification kit, and then is subjected to double enzyme digestion with plasmid pcDNA 3.1-FLAG, and then is connected at 16 ℃. The ligation product was transformed into E.coli DH 5. Alpha. After colony PCR identification, positive clones were subjected to sequencing analysis. The successfully constructed recombinant expression vectors are named as pcDNA-DnaK, pcDNA-GroEL, pcDNA-RpsA, pcDNA-TerD, pcDNA-FHA, pcDNA-RplL and pcDNA-PspA respectively.

The specific operations of double enzyme digestion, connection and transformation are carried out according to the corresponding kit instructions.

Example 3 preparation of DNA vaccine and evaluation of immunoprotection Effect thereof

(I) Nocardia seriolae toxin-attacking hybrid snakehead LD ₅₀ Computing

Culturing Nocardia seriolae wild strain N.seriolae strain ZJ0503, preparing bacterial liquid, sequentially diluting Nocardia seriolae stock solution with sterilized PBS solution by 10 times, setting 7 groups of bacterial liquids with different concentrations, respectively 1 × 10 ³ CFU/mL、1×10 ⁴ CFU/mL、1×10 ⁵ CFU/mL、1×10 ⁶ CFU/mL、1×10 ⁷ CFU/mL、1×10 ⁸ CFU/mL and 1X 10 ⁹ CFU/mL。

Healthy hybrid snakeheads were randomly assigned to 0.1m ³ The breeding barrels are used for breeding, 10 tails of each barrel, and 30 barrels. Respectively sucking 7 groups of Nocardia seriolae bacterial solutions with different concentrations, and infecting the hybrid snakeheads by an intraperitoneal injection mode, wherein 10 tails of each group are 100 mu L of each tail; simultaneously injecting PBS solution with equal dosage as a blank control group; 3 parallel groups were set for each concentration of inoculum and placebo. Counting the death number of each group after continuously observing for 14 days, and using the Nocardia seriolae to infect the hybridized snakehead LD ₅₀ And (4) calculating.

Calculating the semi-Lethal Dose (LD) of Nocardia seriolae infected hybrid snakehead according to the statistical results of the mortality rate of Nocardia seriolae infected hybrid snakehead with different concentrations by using statistical software SPSS 21.0 ₅₀ ) Is 3.31X 10 ⁵ CFU/tail.

(I) preparation of DNA vaccine

Respectively inoculating 7 eukaryotic recombinant expression vector bacteria pcDNA-DnaK/BL21, pcDNA-GroEL/BL21, pcDNA-RpsA/BL21, pcDNA-TerD/BL21, pcDNA-FHA/BL21, pcDNA-RplL/BL21, pcDNA-PspA/BL21 and empty plasmid bacteria pcDNA 3.1-FLAG/BL21 into an ampicillin LB liquid culture medium containing 100 mu g/mL, placing the culture medium in a constant temperature shaking table at 37 ℃ for 200r/min to perform shaking culture for 8-10h until the concentration of bacteria liquid is OD ₆₀₀ When the oscillation reaches 2.0-3.0, the oscillation is stopped. Adding the bacterial liquid into a 50mL centrifuge tube, centrifuging at 8000r/min for 1min by a centrifuge, and collecting thallus (the thallus can be collected by centrifuging for several times when the bacterial liquid is more). E.Z.N.A @ Fastfiler Endo-free Plasmid Maxiprep kit is used for extracting a large amount of endotoxin-free eukaryotic expression recombinant plasmids, and the specific operation refers to the kit specification. The 8 endotoxin-free plasmids were each diluted to a concentration of 250. Mu.g/mL for subsequent immunoblotchy snakehead assay.

(III) DNA vaccine immune hybrid snakehead

Distributing the hybrid snakehead fry to 0.5m ³ The breeding is carried out in 27 barrels, and 50 tails are bred in each barrel. Respectively sucking 100 mu L of endotoxin-free eukaryotic expression recombinant plasmids, immunizing hybrid snakeheads in a dorsal fin intramuscular injection mode, and respectively naming the recombinant plasmids as pcDNA-DnaK, pcDNA-GroEL, pcDNA-RpsA, pcDNA-TerD, pcDNA-FHA, pcDNA-RplL, pcDNA-PspA and empty plasmid pcDNA 3.1-FLAG groups(ii) a Meanwhile, the hybridized snakeheads injected with PBS solution with equal dosage are used as blank control groups. The experimental group and the control group are respectively provided with 3 parallel groups, and each group is injected with 50 hybridized snakeheads.

(IV) detecting GroEL, rpsA, dnaK, terD, FHA, rplL and PspA gene expression in hybrid snakehead

3 hybridized snakeheads are randomly taken from each group for tissue sampling at the 7 th and 35d of the DNA vaccine immune hybridized snakeheads respectively. The tissue samples of the muscles, livers, spleens and head kidneys of hybridized snakehead (the size of the tissue sample is about 0.2cm multiplied by 0.2 cm) are taken by aseptic operation and placed in a 1.5mL centrifuge tube containing 1mL of RNAlater solution, the tube is placed at 4 ℃ overnight to ensure that the RNAlater solution fully fixes the tissue samples, and the tissue samples are preserved at-80 ℃ to be prepared for extracting RNA for RT-PCR detection of target genes of DNA vaccines.

RNA tissue samples (muscle, liver, spleen and head kidney) were extracted using the EasyPure RNA Kit. The RNA of the hybridized snakehead tissue sample is reversely transcribed into cDNA by adopting a TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix reverse transcription kit, and the cDNA is used as an RT-PCR detection template. RT-PCR reaction primers, system and procedure were referred to "example 2".

In order to determine the expression condition of the constructed eukaryotic expression recombinant plasmids of pcDNA-DnaK, pcDNA-GroEL, pcDNA-RpsA, pcDNA-Terd, pcDNA-FHA, pcDNA-RplL and pcDNA-PspA in the tissues of the hybridized snakehead, the muscles, livers, spleens and head kidney tissues of each group of experimental fishes are subjected to RT-PCR detection at 7 th and 35d of the injected recombinant plasmids, and agarose gel electrophoresis can find bands with the sizes consistent with the target genes GroEL, rpsA, dnaK, terd, FHA, rplL and PspA, thereby indicating that the recombinant plasmids are expressed in the tissues of the hybridized snakehead.

(V) serum antibody titer determination

(1) Serum sampling

3 rd hybridized snakeheads are randomly taken from each group for serum sample collection respectively at 0 th, 1 st, 3 th, 5 th, 7 th, 14 th, 21 st, 28 th and 35 th d of hybridized snakeheads immunized by DNA vaccines. A1 mL syringe is rinsed with heparin sodium solution, and about 1mL of blood per hybrid snakehead is extracted in a tail vein blood sampling manner. Removing needle from syringe, squeezing gently, transferring blood to 1.5mL centrifuge tube, standing at room temperature for 2h, standing overnight at 4 deg.C, centrifuging at 4000r/min at 4 deg.C for 5min, sucking supernatant, and storing at-80 deg.C.

(2) Measurement of serum enzyme Activity

5 enzyme activities of a serum sample are measured by adopting Lysozyme (LZM), peroxidase (POD), total superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (ALP) of Nanjing institute of bioengineering, and the specific operation steps refer to corresponding kit instructions. Experimental results the data obtained were analyzed by multiple comparative tests (p >0.05, no significant difference; p <0.05, significant difference; p <0.01, significant difference) and Standard Deviation (SD) using SPSS 21.0 software.

The results show that, compared with the pcDNA 3.1-FLAG group and the PBS control group, the five enzymes of the hybridized snakehead serum are activated at different time points after immunization in 7 DNA vaccine groups, the expression level is increased, and the expression level reaches the peak value at different time points.

(3) Serum antibody titer determination

The indirect ELISA method is adopted to detect the serum antibody titer of the DNA vaccine immune hybrid snakehead, and the calculation formula is as follows:

antibody titer (experimental group T/control group C) = (OD 450 of serum to be tested in experimental group-OD 450 of blank)/(OD 450 of serum to be tested in negative control group-OD 450 of blank).

When T/C >2.1, the dilution of the antiserum was its antibody titer.

The antibody titer of the serum of the hybrid snakehead after the immunization of each group of DNA vaccines (pcDNA-DnaK, pcDNA-GroEL, pcDNA-RpsA, pcDNA-TerD, pcDNA-FHA, pcDNA-RplL and pcDNA-PspA) is measured by an indirect ELISA method for 7, 14, 21, 28 and 35d after the immunization of each group of DNA vaccines respectively. The result shows that after the hybrid snakehead is inoculated with each group of DNA vaccine, specific immune reaction occurs, and the serum antibody titer is obviously increased at different time points. Compared with the pcDNA 3.1-FLAG group and the PBS control group, the generation of the antibody can be detected in the 7 th day after the immunization of the hybridized snakeheads in each DNA vaccine group, and the expression quantity reaches the peak value at different time points (the pcDNA-GroEL group: the peak value: 28d; the titer: 1.

(VI) qRT-PCR detection of hybrid snakehead immune related gene differential expression

(1) 3 hybridized snakeheads are randomly taken for tissue sampling in 0 th, 1 th, 3 th, 5 th, 7 th, 14 th, 21 th, 28 th and 35 th days of DNA vaccine immune hybridized snakeheads respectively. The tissue samples of muscles, livers, spleens and head kidneys of the hybrid snakehead (the size of the tissue sample is about 0.2cm multiplied by 0.2 cm) are taken by aseptic operation and placed in a 1.5mL centrifuge tube containing 1mL of RNAlater solution, the tube is placed at 4 ℃ overnight to ensure that the RNAlater solution fully fixes the tissue samples, and the tissue samples are preserved at-80 ℃ for extracting RNA and being used for qRT-PCR detection of the immune related gene expression test of the hybrid snakehead.

(2) RNA tissue samples (liver, spleen and head kidney) were extracted using the EasyPure RNA Kit. The RNA of the hybridized snakehead tissue sample is reversely transcribed into cDNA by adopting a TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix reverse transcription kit, and the cDNA is used as a fluorescence quantitative qRT-PCR detection template.

(3) Primers are designed according to sequences of hybrid snakehead immune related genes MHCI alpha, MHCII alpha, IL-1 beta, TNF alpha, CD4 and CD8 alpha, and each pair of primers is shown in Table 2. The fluorescence quantitative qRT-PCR detects the relative expression quantity of the immune related gene mRNA, beta-actin is used as an internal reference gene, and a reaction system and an amplification program are as follows:

TABLE 2

The relative expression amounts of the reference gene and the target gene were set to 2 ^(-△△Ct) Calculating a method; the resulting data were tested for multiple comparisons (p) using SPSS 21.0 software>0.05, no significant difference; p is a radical of<0.05, significant difference; p is a radical of<0.01, very significant difference) and Standard Deviation (SD) analysis.

Experiment analysis was performed by fluorescent quantitative qRT-PCR on the expression of 6 immune-related genes (MHCI alpha, MHCII alpha, CD4, CD8 alpha, IL-1 beta and TNF alpha) in hybrid snakehead liver, spleen and head kidney tissues at 9 time points (0, 1, 3, 5, 7, 14, 21, 28, 35 d). The results show that compared with the pcDNA 3.1-FLAG group and the PBS control group, the mRNA expression levels of MHCI alpha, MHCII alpha, CD4, CD8 alpha, IL-1 beta and TNF alpha genes of 7 groups of DNA vaccine hybrid snakeheads are up-regulated in the liver, spleen and kidney of the hybrid snakeheads, and the expression levels reach peak values at different time points, and then a certain reduction phenomenon occurs, but the expression levels are still higher than those of the control group.

(VII) calculation of wild strain toxicity counteracting and immune protection rate

According to LD ₅₀ Calculating results, and preparing a half-lethal concentration bacterial solution of a wild strain N.seroiolae strain ZJ0503 of the hybrid snakehead nocardia seriolae. After each group of DNA vaccines are used for immunizing hybrid snakeheads for 35 days, the bacterial liquid with the semilethal concentration of the Nocardia seriolae wild strain N.seriolae strain ZJ0503 is respectively sucked to carry out the challenge test on the hybrid snakeheads in an intraperitoneal injection mode. The immune groups of pcDNA-DnaK, pcDNA-GroEL, pcDNA-RpsA, pcDNA-TerD, pcDNA-FHA, pcDNA-RplL, pcDNA-PspA and empty plasmid pcDNA 3.1-FLAG and the control group PBS respectively attack 3 groups of hybrid snakeheads, each group has 30 tails, and each tail has 100 muL. The observations were continued for 14d and the death mantissas were recorded.

Carrying out a live bacteria challenge test on wild Nocardia seriolae strain N.seriolae strain ZJ0503 after 35d of Nocardia seriolae DNA vaccine immune hybrid snakehead, wherein the experimental group and the control group of hybrid snakehead have death in different degrees within 14d, the death time is concentrated in 5-10d after live bacteria challenge, and then the experimental group and the control group are basically in a stable state. The dead fish body is clinically identified, and the diseased fish body presents abdominal swelling and has ascites; the cells were dissected and observed to show distinct white nodular structures in the liver, spleen and kidney. Pathogenic separation and identification are carried out on the dead hybrid snakeheads, and the diseased hybrid snakeheads are confirmed to be infected and dead by live bacteria of wild Nocardia seriolae strain ZJ0503 injected. In addition, the common antigen DNA vaccine of three pathogenic bacteria of the fish nocardiosis has certain immune protection effect on preventing and treating the fish nocardiosis by the hybrid snakehead, and the immune protection rate is respectively pcDNA-PspA group: 57.83 percent; pcDNA-FHA group: 62.64 percent; pcDNA-DnaK group: 53.01 percent; pcDNA-RplL group: 78.31 percent; pcDNA-TerD group: 83.14 percent; pcDNA-RpsA group: 71.08 percent; pcDNA-GroEL group: 80.71% (Table 3).

TABLE 3

* Relative immune protection Rate (RPS) = {1- [ immune group mortality (%)/control group mortality (%) ] } × 100%

N.a. = not applicable

As can be seen from Table 3, the DNA vaccine prepared by the invention has different degrees of prevention and treatment effects on fish Nocardia diseases.

Those skilled in the art will appreciate that the above embodiments are merely exemplary embodiments and that various changes, substitutions, and alterations can be made without departing from the spirit and scope of the invention.

Sequence listing

<110> Shenzhen institute of Guangdong ocean university; guangdong university of oceans; shenzhen Zhenhai Biotech Co Ltd

<120> fish Nocardia disease common antigen DNA vaccine, preparation and application thereof

<130> HP191210LZ

<160> 42

<170> PatentIn version 3.3

<210> 1

<211> 29

<212> DNA

<213> Artificial sequence

<400> 1

cgggatccat gcccatccct gatctggag 29

<210> 2

<211> 29

<212> DNA

<213> Artificial sequence

<400> 2

ggaattccta gaagtccatg ccgcccatg 29

<210> 3

<211> 28

<212> DNA

<213> Artificial sequence

<400> 3

cgggatccat gcccaccact gtcacctc 28

<210> 4

<211> 29

<212> DNA

<213> Artificial sequence

<400> 4

ggaattctca ggcgttgccc gacagcttc 29

<210> 5

<211> 29

<212> DNA

<213> Artificial sequence

<400> 5

gaagatctat ggctcgtgcg gtcggtatc 29

<210> 6

<211> 37

<212> DNA

<213> Artificial sequence

<400> 6

ggaattctca cttcttctca ggctcctcga cgacctc 37

<210> 7

<211> 29

<212> DNA

<213> Artificial sequence

<400> 7

cgggatccat ggccaacgtt gatgagctg 29

<210> 8

<211> 28

<212> DNA

<213> Artificial sequence

<400> 8

ggaattctta cttgacggtg atcttggc 28

<210> 9

<211> 29

<212> DNA

<213> Artificial sequence

<400> 9

gaagatctat ggctaatccg ttcgtgaag 29

<210> 10

<211> 27

<212> DNA

<213> Artificial sequence

<400> 10

ggaattctta ctgctgggcg gcctggc 27

<210> 11

<211> 29

<212> DNA

<213> Artificial sequence

<400> 11

cgggatccat gggtgtcagt ttgtccaag 29

<210> 12

<211> 28

<212> DNA

<213> Artificial sequence

<400> 12

ccgctcgagt cagacgttga cgccgaag 28

<210> 13

<211> 27

<212> DNA

<213> Artificial sequence

<400> 13

cgggatccat gagcgagaac aaggacc 27

<210> 14

<211> 29

<212> DNA

<213> Artificial sequence

<400> 14

ccgctcgagc tatagagccg aatcgctcg 29

<210> 15

<211> 25

<212> DNA

<213> Artificial sequence

<400> 15

tgcactcatg gaaggcattt tacac 25

<210> 16

<211> 19

<212> DNA

<213> Artificial sequence

<400> 16

gggtagcctc tgagaatgt 19

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence

<400> 17

actttggtac gcggacttca 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence

<400> 18

gaactttggt acgcggactt 42

<210> 19

<211> 22

<212> DNA

<213> Artificial sequence

<400> 19

aatctgtctt ctgacctcca ac 22

<210> 20

<211> 19

<212> DNA

<213> Artificial sequence

<400> 20

cacccatttt ccgctatct 19

<210> 21

<211> 18

<212> DNA

<213> Artificial sequence

<400> 21

tggtcggttt ccttggtt 18

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence

<400> 22

ctttgtgcat gaatccccat 20

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence

<400> 23

gacaaaagca tcctgacgac 20

<210> 24

<211> 18

<212> DNA

<213> Artificial sequence

<400> 24

gaaaattggc ggacctga 18

<210> 25

<211> 22

<212> DNA

<213> Artificial sequence

<400> 25

ccgttttaca ccggatactt tg 22

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence

<400> 26

tactcgccct tcatcaccac 20

<210> 27

<211> 18

<212> DNA

<213> Artificial sequence

<400> 27

atgtcgccct ggacttcg 18

<210> 28

<211> 18

<212> DNA

<213> Artificial sequence

<400> 28

ctgggcaacg gaacctct 18

<210> 29

<211> 1656

<212> DNA

<213> Nocardiaceae

<400> 29

atgcccatcc ctgatctgga ggatctcaac gcaatggcca agacaattgc gtacgacgaa 60

gaggctcgcc gcggactcga gcgtggcctg aacagcctcg ccgacgctgt caaggtgacc 120

cttggcccca agggccgcaa tgtcgtgctg gagaagaagt ggggcgcccc caccatcacc 180

aacgatggtg tctccatcgc gaaggaaatc gagctcgagg acccgtacga gaagatcggc 240

gcggagctcg tcaaggaagt tgcgaagaag accgacgacg tcgcgggtga cggcaccacc 300

accgccaccg tgctggccca ggcgctggtc cgcgagggcc tgcgcaacgt ggccgccggc 360

gccaacccgc tcggcctgaa gcgcggcatc gagaaggccg tcgaggccgt caccgcctcg 420

ctgctcgaca ccgccaagga ggtcgagacc aaggagcaga tcgccgccac cgccggtatc 480

tcggccggcg actcgtccat cggtgagctc atcgccgagg ccatggacaa ggtcggcaag 540

gaaggcgtca tcaccgtcga ggagagcaac accttcggtc tccagctgga gctgaccgag 600

ggcatgcgct tcgacaaggg ctacatctcc ggttacttcg tgaccgaccc ggagcgtcag 660

gaagcggtcc tcgaggaccc gtacatcctg ctggtcggct cgaagatctc gaccgtcaag 720

gacctgctgc cgctgctgga gaaggtcatc caggccggca agccgctgct gatcatcgcc 780

gaagacgttg agggcgaagc gctctcgacc ctggtcgtga acaagatccg cggcaccttc 840

aagtccatcg ccgtcaaggc cccgggcttc ggcgaccgcc gcaaggccat gctggccgac 900

atcgccatcc tgaccggtgg cgaggtcatc agcgaagagg tcggcctctc tctcgagacc 960

gccggtctgg agctgctggg caccgcccgc aaggtcgtca tcaccaagga cgagaccacc 1020

atcgtcgagg gcgcgggcga cccggaggcc atcaagggcc gcgtggcgca gatccgcacc 1080

gagatcgaga actcggactc ggactacgac cgtgagaagc tgcaggagcg cctggccaag 1140

ctggccggtg gcgttgctgt catcaaggcg ggcgcggcca ccgaggtcga gctcaaggag 1200

cgcaagcacc gcatcgagga cgccgtccgc aacgcgaagg ccgccgtcga agagggcatc 1260

gtcgccggtg gtggcgtggc cctgctgcag gcctccccgg ccctggacgc gctgtccctg 1320

accggtgacg aggccaccgg cgcgaacatc gttcgcgtcg cgctgtccgc gccgctgaag 1380

cagatcgcct tcaacgcggg cctggagccc ggcgtcgtcg ccgagaaggt ctcgaacctc 1440

ccggccggcc acggcctgaa cgccgagtcc ggcgagtacg tcgacctgct ggccgccggc 1500

gttgccgacc cggtcaaggt cacccgctcg gccctgcaga acgcggcctc catcgcggcc 1560

ctgttcctca ccaccgaggc cgtcgtcgcc gacaagccgg agaaggccgc ccccgcgggc 1620

gacccgaccg gcggcatggg cggcatggac ttctag 1656

<210> 30

<211> 468

<212> DNA

<213> Nocardiaceae

<400> 30

atgagcgaga acaaggaccc gggttaccag gaaacggcag cagagaccac ttcggtgttc 60

cgtgccgact tcctgaacga ggtcgacacg tcgcgccccg agcagacagg cgagcagccg 120

gtacagggag tcgagggtct gcccgcgggc gcggccctgc tggtggtcaa gcgtggtccg 180

aacgccggtt cgcgattcct gctcgaccag ccgaccacct cggcgggccg ccaccccgac 240

agcgacatct tcctggacga tgtcaccgtc agccgtcgcc acgcggaatt ccgccaggac 300

gacgacacct tccaggtggt cgatgtgggc agcctcaacg gcacctacgt caaccgggag 360

ccggtggatt cctcggaact gcagaacggt gacgaggtcc agatcggcaa gttccgcctg 420

gtcttcctga ccggccccaa gccggtttcg agcgattcgg ctctatag 468

<210> 31

<211> 831

<212> DNA

<213> Nocardiaceae

<400> 31

atggctaatc cgttcgtgaa ggcctggaag tacctgatgg ccctcttcga ctcgaagatc 60

gaggagcatg cggatccgaa ggttcagatc cagcaggcca tcgaggaagc ccagcgccag 120

catcaggccc tgtcgcagca ggccgcgtcg gtgatcggca accagcgcca gctggagatg 180

aagctgaacc gccagctgga cgaggtcgag aagctcaatg ccaatgcgcg ccaggcggtt 240

atgctcgccg accaggccag cggcgcgggt gacaccgaga aggcgatcca gtacaccaat 300

gccgcagagg ctttcgccgc gcagctggtg accgccgagc agtccgtcga ggatctgaag 360

gtgctgcacg accagtcgct gcaggccgcc gcgcaggcca agaaggccgt cgagcagaac 420

gccatgctgc tgcagcagaa ggtcgccgag cgcaccaagc tgctttccca gctggagcag 480

gccaagatgc aggagcaggt ctcggcctcg ctgcagcaga tggattccac gctgtccgcg 540

cccggtgcgg tccccagcct ggacgcggta cgcgagaaga tcgagcgccg ctacgccaac 600

gccctgggtg ccgccgagct ggcgggcaac tcggtgcagg gccgcatgct cgaggtccag 660

caggccagcg tccagatggc cggccacaac cgcctcgagc agatccgcgc ctccatgcgc 720

ggcgacgccc tccccgccgg tggcgccgcc cagccgcaga tccagcaggg ccagcccgcc 780

cagcccgccg cccagcccaa cttcaacaag ggccaggccg cccagcagta a 831

<210> 32

<211> 1467

<212> DNA

<213> Nocardiaceae

<400> 32

atgcccacca ctgtcacctc gccgcaggtt gccgtcaacg acatcggcac tgccgaggac 60

ttcctcgccg ccatcgacaa gaccatcaag tacttcaacg acggtgacat cgtcgaagga 120

accatcgtca aggtcgatcg cgatgaggtc cttctcgaca tcggttacaa gaccgaaggc 180

gtgatcccgt ctcgcgaact gtccatcaag cacgatgtcg acccggccga ggtcgtttcc 240

gtgggcgatg aggtcgaggc cctcgttctc accaaggagg acaaggaagg ccgcctgatc 300

ctgtcgaaga agcgcgctca gtacgagcgg gcttggggca cgatcgagga gctcaaggag 360

aaggacgagg ccgtcaaggg caccgtcatc gaggtcgtca agggcggcct gatcctggac 420

atcggtctgc gcggcttcct cccggcttcg ctggtcgaga tgcgccgtgt ccgcgacctc 480

cagccgtacg tcggcaagga gatcgaggcc aagatcatcg agctcgacaa gaaccgcaac 540

aacgtggtcc tgtcgcgtcg cgcctggctc gagcagaccc agtccgaggt ccgcagcgag 600

ttcctgcacc agctccagaa gggccaggtc cgcaagggcg tcgtgtcctc catcgtcaac 660

ttcggtgcct tcgtggacct gggtggcgtc gacggtctgg tgcacgtctc cgagctgtcc 720

tggaagcaca tcgaccaccc gtccgaggtt gtcgaggtcg gcaacgaggt caccgtcgag 780

gttctcgacg tcgatctcga ccgcgagcgt gtctccctgt cgctcaaggc gacccaggaa 840

gacccgtggc gtcagttcgc ccgcacccac gcgatcggtc agatcgggcc gggcaaggtc 900

accaagctgg ttccgttcgg tgcgttcgtg cgcgtcgaag agggcatcga gggcctggtt 960

cacatctccg agctggccga gcgccacgtc gaggtcccgg accaggttgt cgccgtcggc 1020

gacgacgcca tggtcaaggt catcgacatc gacctcgagc gtcgccggat ctcgctgtcg 1080

ctgaagcagg ccaacgagga ctacaccgcc gagttcgacc cgtcgaagta cggcatggcc 1140

gacagctacg acgaccaggg caactacatc ttccccgagg gcttcgactc cgagaccaac 1200

gaatggctcg agggcttcga gaagcagcgt gaagagtggg agacccggta cgccgaggcc 1260

gagcgtcgcc acaagatgca caccgctcag atggagaaga tggcggccga cgccgccgct 1320

gaggccgcca acggcgggcc gcagaactac tcctcggagt ccggttcgca ggctgccgct 1380

tcgtcctcct ccgagtcggc cggtggctcg ctcgcgagcg acgcgcagtt ggctgccctg 1440

cgtgagaagc tgtcgggcaa cgcctga 1467

<210> 33

<211> 576

<212> DNA

<213> Nocardiaceae

<400> 33

atgggtgtca gtttgtccaa gggcggtaac gtttcactga ccaagcaggc tccgaacctg 60

acccaggtgg ccgtcggcct cggctgggac attcggacca ccaccggcac cgacttcgac 120

ctcgacgcca gcgccatcgc caccggcgcc gacaagaagg cgctgtccga caagcacttc 180

gtcttcttca acaacctgca gtcccccgag ggcaccatcg tgcacaccgg cgacaacctc 240

accggtgagg gcgagggcga cgacgaggtc atcaatatcg acctggccaa caccccgccg 300

gccatcgagt ccatcttctt cccggtctcg atctacgacg cggactcccg cggccagagc 360

ttcggccagg tccgcaatgc ctacatccgc gtggtggacc gcgccaacgg tgccgagctg 420

gcacgctacg acctgtccga ggacgcctct accgaaaccg ccatggtgtt cggcgagctc 480

taccgcaaca atggtgagtg gaagttccgc gccgtcggtc agggttacgc gtccggtctc 540

gcgggcatcg cccgcgactt cggcgtcaac gtctga 576

<210> 34

<211> 1839

<212> DNA

<213> Nocardiaceae

<400> 34

atggctcgtg cggtcggtat cgacctcggg accacgaact ctgtcatcgc cgttctcgaa 60

ggcggcgagc cggtcgtcgt ggccaactcg gaaggatcgc ggaccactcc gtcgatcgtc 120

gcgttcgcga agaacggcga ggtgctcgtc ggtcagcccg cgaagaacca ggccgtcacc 180

aacgtcgacc gcaccattcg gtccgtcaag cgccacatgg gcgaggactg gtcggtcgag 240

atcgacggca agaagtacac cccgcaggag atttccgcgc gcacgctcat gaagctgaag 300

cgcgacgccg aggcctacct cggtgaggag atcaccgacg cggtcatcac cgtccccgcc 360

tacttcgagg acgcgcagcg ccaggccacc aaggaggccg ggcagatcgc gggcctgaac 420

gtcctgcgca tcgtcaacga gccgaccgcg gccgcgctgg cctacggcct ggacaagggc 480

gacaaggaac agaccatcct ggtcttcgac ctcggcggcg gcaccttcga cgtgtccctg 540

ctcgagatcg gcgagggcgt cgtcgaggtc cgcgcgacct ccggtgacaa ccacctcggt 600

ggcgacgact gggacgagcg catcgtctcc tggctggtcg acaagttccg gggcacctcg 660

ggcatcgacc tgaccaagga caagatggcc atgcagcgtc tgcgcgaggc cgccgagaag 720

gcgaagatcg agctgagctc ctcgcagagc acctcgatca acctgcccta catcacggtc 780

gacgcggaca agaacccgct gttcctcgac gagcagctga cccgcgccga gttccagaag 840

atcacctccg atctgctgga ccgcacccgc aagccgttcc agtcggtcat caaggacgcc 900

ggtatctccg tcggcgacat cgaccacgtt gtgctcgttg gtggttcgac ccgtatgccc 960

gccgtgtccg acctggtcaa ggaactgacc ggcggcaagg agcccaacaa gggcgtgaac 1020

ccggacgagg tcgtcgccgt cggcgccgcc ctgcaggccg gtgtgctcaa gggtgaggtc 1080

aaggacgtcc tgctgctcga tgtcaccccg ctgtcgctgg gcatcgagac caagggcggc 1140

gtgatgacca agctcatcga gcgcaacacc acgatcccga ccaagcgttc ggagaccttc 1200

accacggccg acgacaacca gccgtccgtg cagatccagg tcttccaggg tgagcgtgag 1260

atcgcctcgc acaacaagct gctcggatcc ttcgagctga ccggcatccc gccggccccg 1320

cgcggcgtgc cgcagatcga ggtcaccttc gacatcgacg ccaacggcat cgtccacgtg 1380

acggccaagg acaagggcac cggcaaggag aacacgatca agatccagga cggctccggc 1440

ctgtccaagg aggagatcga ccgcatggtc cgcgacgccg aggcgcacgc cgccgaggac 1500

aaggagcggc gcgaggaggc ggagacccgc aaccaggccg agtcgctggt gcaccagacc 1560

gagaagttca tcaaggacaa cgaggacaag gtcccggccg agatcaagac caaggtcgag 1620

gcggccatcg ccgacgccaa cgaggcgctc gagggcaccg acatcgcggc catcaagacc 1680

gcggtggaga agctcgccac cgagtcgcag gcgctgggcc aggccatcta cgaggcctcg 1740

gccgcggatc aggccgccca gggcaacggt gctgcggcgc cgcagtccga tgacaccgtg 1800

gtggacgccg aggtcgtcga ggagcctgag aagaagtga 1839

<210> 35

<211> 381

<212> DNA

<213> Nocardiaceae

<400> 35

atggccaacg ttgatgagct gctcgagacc ttcgccggca tgaccctgct ggagctgtcc 60

gagttcgtga aggcgttcga ggacaagttc gaggtcaccg ctgccgctcc ggtcgccgtc 120

gccgtcgcgg gtggcgctgc tgccccggcc gaggccgccg aggagcagga cgagttcgac 180

gtcatcctcg agggtgccgg cgacaagaag atccaggtca tcaaggtcgt gcgtgagatc 240

gtctccggcc tgggcctgaa ggaagccaag gacctggtcg agggcgcccc gaagccgatc 300

ctggagaagg tcgccaagga ggccgccgag gccgccaagg cgaagctgga agaggccggc 360

gccaagatca ccgtcaagta a 381

<210> 36

<211> 551

<212> PRT

<213> Nocardiaceae

<400> 36

Met Pro Ile Pro Asp Leu Glu Asp Leu Asn Ala Met Ala Lys Thr Ile

1 5 10 15

Ala Tyr Asp Glu Glu Ala Arg Arg Gly Leu Glu Arg Gly Leu Asn Ser

20 25 30

Leu Ala Asp Ala Val Lys Val Thr Leu Gly Pro Lys Gly Arg Asn Val

35 40 45

Val Leu Glu Lys Lys Trp Gly Ala Pro Thr Ile Thr Asn Asp Gly Val

50 55 60

Ser Ile Ala Lys Glu Ile Glu Leu Glu Asp Pro Tyr Glu Lys Ile Gly

65 70 75 80

Ala Glu Leu Val Lys Glu Val Ala Lys Lys Thr Asp Asp Val Ala Gly

85 90 95

Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gln Ala Leu Val Arg Glu

100 105 110

Gly Leu Arg Asn Val Ala Ala Gly Ala Asn Pro Leu Gly Leu Lys Arg

115 120 125

Gly Ile Glu Lys Ala Val Glu Ala Val Thr Ala Ser Leu Leu Asp Thr

130 135 140

Ala Lys Glu Val Glu Thr Lys Glu Gln Ile Ala Ala Thr Ala Gly Ile

145 150 155 160

Ser Ala Gly Asp Ser Ser Ile Gly Glu Leu Ile Ala Glu Ala Met Asp

165 170 175

Lys Val Gly Lys Glu Gly Val Ile Thr Val Glu Glu Ser Asn Thr Phe

180 185 190

Gly Leu Gln Leu Glu Leu Thr Glu Gly Met Arg Phe Asp Lys Gly Tyr

195 200 205

Ile Ser Gly Tyr Phe Val Thr Asp Pro Glu Arg Gln Glu Ala Val Leu

210 215 220

Glu Asp Pro Tyr Ile Leu Leu Val Gly Ser Lys Ile Ser Thr Val Lys

225 230 235 240

Asp Leu Leu Pro Leu Leu Glu Lys Val Ile Gln Ala Gly Lys Pro Leu

245 250 255

Leu Ile Ile Ala Glu Asp Val Glu Gly Glu Ala Leu Ser Thr Leu Val

260 265 270

Val Asn Lys Ile Arg Gly Thr Phe Lys Ser Ile Ala Val Lys Ala Pro

275 280 285

Gly Phe Gly Asp Arg Arg Lys Ala Met Leu Ala Asp Ile Ala Ile Leu

290 295 300

Thr Gly Gly Glu Val Ile Ser Glu Glu Val Gly Leu Ser Leu Glu Thr

305 310 315 320

Ala Gly Leu Glu Leu Leu Gly Thr Ala Arg Lys Val Val Ile Thr Lys

325 330 335

Asp Glu Thr Thr Ile Val Glu Gly Ala Gly Asp Pro Glu Ala Ile Lys

340 345 350

Gly Arg Val Ala Gln Ile Arg Thr Glu Ile Glu Asn Ser Asp Ser Asp

355 360 365

Tyr Asp Arg Glu Lys Leu Gln Glu Arg Leu Ala Lys Leu Ala Gly Gly

370 375 380

Val Ala Val Ile Lys Ala Gly Ala Ala Thr Glu Val Glu Leu Lys Glu

385 390 395 400

Arg Lys His Arg Ile Glu Asp Ala Val Arg Asn Ala Lys Ala Ala Val

405 410 415

Glu Glu Gly Ile Val Ala Gly Gly Gly Val Ala Leu Leu Gln Ala Ser

420 425 430

Pro Ala Leu Asp Ala Leu Ser Leu Thr Gly Asp Glu Ala Thr Gly Ala

435 440 445

Asn Ile Val Arg Val Ala Leu Ser Ala Pro Leu Lys Gln Ile Ala Phe

450 455 460

Asn Ala Gly Leu Glu Pro Gly Val Val Ala Glu Lys Val Ser Asn Leu

465 470 475 480

Pro Ala Gly His Gly Leu Asn Ala Glu Ser Gly Glu Tyr Val Asp Leu

485 490 495

Leu Ala Ala Gly Val Ala Asp Pro Val Lys Val Thr Arg Ser Ala Leu

500 505 510

Gln Asn Ala Ala Ser Ile Ala Ala Leu Phe Leu Thr Thr Glu Ala Val

515 520 525

Val Ala Asp Lys Pro Glu Lys Ala Ala Pro Ala Gly Asp Pro Thr Gly

530 535 540

Gly Met Gly Gly Met Asp Phe

545 550

<210> 37

<211> 155

<212> PRT

<213> Nocardiaceae

<400> 37

Met Ser Glu Asn Lys Asp Pro Gly Tyr Gln Glu Thr Ala Ala Glu Thr

1 5 10 15

Thr Ser Val Phe Arg Ala Asp Phe Leu Asn Glu Val Asp Thr Ser Arg

20 25 30

Pro Glu Gln Thr Gly Glu Gln Pro Val Gln Gly Val Glu Gly Leu Pro

35 40 45

Ala Gly Ala Ala Leu Leu Val Val Lys Arg Gly Pro Asn Ala Gly Ser

50 55 60

Arg Phe Leu Leu Asp Gln Pro Thr Thr Ser Ala Gly Arg His Pro Asp

65 70 75 80

Ser Asp Ile Phe Leu Asp Asp Val Thr Val Ser Arg Arg His Ala Glu

85 90 95

Phe Arg Gln Asp Asp Asp Thr Phe Gln Val Val Asp Val Gly Ser Leu

100 105 110

Asn Gly Thr Tyr Val Asn Arg Glu Pro Val Asp Ser Ser Glu Leu Gln

115 120 125

Asn Gly Asp Glu Val Gln Ile Gly Lys Phe Arg Leu Val Phe Leu Thr

130 135 140

Gly Pro Lys Pro Val Ser Ser Asp Ser Ala Leu

145 150 155

<210> 38

<211> 276

<212> PRT

<213> Nocardiaceae

<400> 38

Met Ala Asn Pro Phe Val Lys Ala Trp Lys Tyr Leu Met Ala Leu Phe

1 5 10 15

Asp Ser Lys Ile Glu Glu His Ala Asp Pro Lys Val Gln Ile Gln Gln

20 25 30

Ala Ile Glu Glu Ala Gln Arg Gln His Gln Ala Leu Ser Gln Gln Ala

35 40 45

Ala Ser Val Ile Gly Asn Gln Arg Gln Leu Glu Met Lys Leu Asn Arg

50 55 60

Gln Leu Asp Glu Val Glu Lys Leu Asn Ala Asn Ala Arg Gln Ala Val

65 70 75 80

Met Leu Ala Asp Gln Ala Ser Gly Ala Gly Asp Thr Glu Lys Ala Ile

85 90 95

Gln Tyr Thr Asn Ala Ala Glu Ala Phe Ala Ala Gln Leu Val Thr Ala

100 105 110

Glu Gln Ser Val Glu Asp Leu Lys Val Leu His Asp Gln Ser Leu Gln

115 120 125

Ala Ala Ala Gln Ala Lys Lys Ala Val Glu Gln Asn Ala Met Leu Leu

130 135 140

Gln Gln Lys Val Ala Glu Arg Thr Lys Leu Leu Ser Gln Leu Glu Gln

145 150 155 160

Ala Lys Met Gln Glu Gln Val Ser Ala Ser Leu Gln Gln Met Asp Ser

165 170 175

Thr Leu Ser Ala Pro Gly Ala Val Pro Ser Leu Asp Ala Val Arg Glu

180 185 190

Lys Ile Glu Arg Arg Tyr Ala Asn Ala Leu Gly Ala Ala Glu Leu Ala

195 200 205

Gly Asn Ser Val Gln Gly Arg Met Leu Glu Val Gln Gln Ala Ser Val

210 215 220

Gln Met Ala Gly His Asn Arg Leu Glu Gln Ile Arg Ala Ser Met Arg

225 230 235 240

Gly Asp Ala Leu Pro Ala Gly Gly Ala Ala Gln Pro Gln Ile Gln Gln

245 250 255

Gly Gln Pro Ala Gln Pro Ala Ala Gln Pro Asn Phe Asn Lys Gly Gln

260 265 270

Ala Ala Gln Gln

275

<210> 39

<211> 488

<212> PRT

<213> Nocardiaceae

<400> 39

Met Pro Thr Thr Val Thr Ser Pro Gln Val Ala Val Asn Asp Ile Gly

1 5 10 15

Thr Ala Glu Asp Phe Leu Ala Ala Ile Asp Lys Thr Ile Lys Tyr Phe

20 25 30

Asn Asp Gly Asp Ile Val Glu Gly Thr Ile Val Lys Val Asp Arg Asp

35 40 45

Glu Val Leu Leu Asp Ile Gly Tyr Lys Thr Glu Gly Val Ile Pro Ser

50 55 60

Arg Glu Leu Ser Ile Lys His Asp Val Asp Pro Ala Glu Val Val Ser

65 70 75 80

Val Gly Asp Glu Val Glu Ala Leu Val Leu Thr Lys Glu Asp Lys Glu

85 90 95

Gly Arg Leu Ile Leu Ser Lys Lys Arg Ala Gln Tyr Glu Arg Ala Trp

100 105 110

Gly Thr Ile Glu Glu Leu Lys Glu Lys Asp Glu Ala Val Lys Gly Thr

115 120 125

Val Ile Glu Val Val Lys Gly Gly Leu Ile Leu Asp Ile Gly Leu Arg

130 135 140

Gly Phe Leu Pro Ala Ser Leu Val Glu Met Arg Arg Val Arg Asp Leu

145 150 155 160

Gln Pro Tyr Val Gly Lys Glu Ile Glu Ala Lys Ile Ile Glu Leu Asp

165 170 175

Lys Asn Arg Asn Asn Val Val Leu Ser Arg Arg Ala Trp Leu Glu Gln

180 185 190

Thr Gln Ser Glu Val Arg Ser Glu Phe Leu His Gln Leu Gln Lys Gly

195 200 205

Gln Val Arg Lys Gly Val Val Ser Ser Ile Val Asn Phe Gly Ala Phe

210 215 220

Val Asp Leu Gly Gly Val Asp Gly Leu Val His Val Ser Glu Leu Ser

225 230 235 240

Trp Lys His Ile Asp His Pro Ser Glu Val Val Glu Val Gly Asn Glu

245 250 255

Val Thr Val Glu Val Leu Asp Val Asp Leu Asp Arg Glu Arg Val Ser

260 265 270

Leu Ser Leu Lys Ala Thr Gln Glu Asp Pro Trp Arg Gln Phe Ala Arg

275 280 285

Thr His Ala Ile Gly Gln Ile Gly Pro Gly Lys Val Thr Lys Leu Val

290 295 300

Pro Phe Gly Ala Phe Val Arg Val Glu Glu Gly Ile Glu Gly Leu Val

305 310 315 320

His Ile Ser Glu Leu Ala Glu Arg His Val Glu Val Pro Asp Gln Val

325 330 335

Val Ala Val Gly Asp Asp Ala Met Val Lys Val Ile Asp Ile Asp Leu

340 345 350

Glu Arg Arg Arg Ile Ser Leu Ser Leu Lys Gln Ala Asn Glu Asp Tyr

355 360 365

Thr Ala Glu Phe Asp Pro Ser Lys Tyr Gly Met Ala Asp Ser Tyr Asp

370 375 380

Asp Gln Gly Asn Tyr Ile Phe Pro Glu Gly Phe Asp Ser Glu Thr Asn

385 390 395 400

Glu Trp Leu Glu Gly Phe Glu Lys Gln Arg Glu Glu Trp Glu Thr Arg

405 410 415

Tyr Ala Glu Ala Glu Arg Arg His Lys Met His Thr Ala Gln Met Glu

420 425 430

Lys Met Ala Ala Asp Ala Ala Ala Glu Ala Ala Asn Gly Gly Pro Gln

435 440 445

Asn Tyr Ser Ser Glu Ser Gly Ser Gln Ala Ala Ala Ser Ser Ser Ser

450 455 460

Glu Ser Ala Gly Gly Ser Leu Ala Ser Asp Ala Gln Leu Ala Ala Leu

465 470 475 480

Arg Glu Lys Leu Ser Gly Asn Ala

485

<210> 40

<211> 191

<212> PRT

<213> Nocardiaceae

<400> 40

Met Gly Val Ser Leu Ser Lys Gly Gly Asn Val Ser Leu Thr Lys Gln

1 5 10 15

Ala Pro Asn Leu Thr Gln Val Ala Val Gly Leu Gly Trp Asp Ile Arg

20 25 30

Thr Thr Thr Gly Thr Asp Phe Asp Leu Asp Ala Ser Ala Ile Ala Thr

35 40 45

Gly Ala Asp Lys Lys Ala Leu Ser Asp Lys His Phe Val Phe Phe Asn

50 55 60

Asn Leu Gln Ser Pro Glu Gly Thr Ile Val His Thr Gly Asp Asn Leu

65 70 75 80

Thr Gly Glu Gly Glu Gly Asp Asp Glu Val Ile Asn Ile Asp Leu Ala

85 90 95

Asn Thr Pro Pro Ala Ile Glu Ser Ile Phe Phe Pro Val Ser Ile Tyr

100 105 110

Asp Ala Asp Ser Arg Gly Gln Ser Phe Gly Gln Val Arg Asn Ala Tyr

115 120 125

Ile Arg Val Val Asp Arg Ala Asn Gly Ala Glu Leu Ala Arg Tyr Asp

130 135 140

Leu Ser Glu Asp Ala Ser Thr Glu Thr Ala Met Val Phe Gly Glu Leu

145 150 155 160

Tyr Arg Asn Asn Gly Glu Trp Lys Phe Arg Ala Val Gly Gln Gly Tyr

165 170 175

Ala Ser Gly Leu Ala Gly Ile Ala Arg Asp Phe Gly Val Asn Val

180 185 190

<210> 41

<211> 612

<212> PRT

<213> Nocardiaceae

<400> 41

Met Ala Arg Ala Val Gly Ile Asp Leu Gly Thr Thr Asn Ser Val Ile

1 5 10 15

Ala Val Leu Glu Gly Gly Glu Pro Val Val Val Ala Asn Ser Glu Gly

20 25 30

Ser Arg Thr Thr Pro Ser Ile Val Ala Phe Ala Lys Asn Gly Glu Val

35 40 45

Leu Val Gly Gln Pro Ala Lys Asn Gln Ala Val Thr Asn Val Asp Arg

50 55 60

Thr Ile Arg Ser Val Lys Arg His Met Gly Glu Asp Trp Ser Val Glu

65 70 75 80

Ile Asp Gly Lys Lys Tyr Thr Pro Gln Glu Ile Ser Ala Arg Thr Leu

85 90 95

Met Lys Leu Lys Arg Asp Ala Glu Ala Tyr Leu Gly Glu Glu Ile Thr

100 105 110

Asp Ala Val Ile Thr Val Pro Ala Tyr Phe Glu Asp Ala Gln Arg Gln

115 120 125

Ala Thr Lys Glu Ala Gly Gln Ile Ala Gly Leu Asn Val Leu Arg Ile

130 135 140

Val Asn Glu Pro Thr Ala Ala Ala Leu Ala Tyr Gly Leu Asp Lys Gly

145 150 155 160

Asp Lys Glu Gln Thr Ile Leu Val Phe Asp Leu Gly Gly Gly Thr Phe

165 170 175

Asp Val Ser Leu Leu Glu Ile Gly Glu Gly Val Val Glu Val Arg Ala

180 185 190

Thr Ser Gly Asp Asn His Leu Gly Gly Asp Asp Trp Asp Glu Arg Ile

195 200 205

Val Ser Trp Leu Val Asp Lys Phe Arg Gly Thr Ser Gly Ile Asp Leu

210 215 220

Thr Lys Asp Lys Met Ala Met Gln Arg Leu Arg Glu Ala Ala Glu Lys

225 230 235 240

Ala Lys Ile Glu Leu Ser Ser Ser Gln Ser Thr Ser Ile Asn Leu Pro

245 250 255

Tyr Ile Thr Val Asp Ala Asp Lys Asn Pro Leu Phe Leu Asp Glu Gln

260 265 270

Leu Thr Arg Ala Glu Phe Gln Lys Ile Thr Ser Asp Leu Leu Asp Arg

275 280 285

Thr Arg Lys Pro Phe Gln Ser Val Ile Lys Asp Ala Gly Ile Ser Val

290 295 300

Gly Asp Ile Asp His Val Val Leu Val Gly Gly Ser Thr Arg Met Pro

305 310 315 320

Ala Val Ser Asp Leu Val Lys Glu Leu Thr Gly Gly Lys Glu Pro Asn

325 330 335

Lys Gly Val Asn Pro Asp Glu Val Val Ala Val Gly Ala Ala Leu Gln

340 345 350

Ala Gly Val Leu Lys Gly Glu Val Lys Asp Val Leu Leu Leu Asp Val

355 360 365

Thr Pro Leu Ser Leu Gly Ile Glu Thr Lys Gly Gly Val Met Thr Lys

370 375 380

Leu Ile Glu Arg Asn Thr Thr Ile Pro Thr Lys Arg Ser Glu Thr Phe

385 390 395 400

Thr Thr Ala Asp Asp Asn Gln Pro Ser Val Gln Ile Gln Val Phe Gln

405 410 415

Gly Glu Arg Glu Ile Ala Ser His Asn Lys Leu Leu Gly Ser Phe Glu

420 425 430

Leu Thr Gly Ile Pro Pro Ala Pro Arg Gly Val Pro Gln Ile Glu Val

435 440 445

Thr Phe Asp Ile Asp Ala Asn Gly Ile Val His Val Thr Ala Lys Asp

450 455 460

Lys Gly Thr Gly Lys Glu Asn Thr Ile Lys Ile Gln Asp Gly Ser Gly

465 470 475 480

Leu Ser Lys Glu Glu Ile Asp Arg Met Val Arg Asp Ala Glu Ala His

485 490 495

Ala Ala Glu Asp Lys Glu Arg Arg Glu Glu Ala Glu Thr Arg Asn Gln

500 505 510

Ala Glu Ser Leu Val His Gln Thr Glu Lys Phe Ile Lys Asp Asn Glu

515 520 525

Asp Lys Val Pro Ala Glu Ile Lys Thr Lys Val Glu Ala Ala Ile Ala

530 535 540

Asp Ala Asn Glu Ala Leu Glu Gly Thr Asp Ile Ala Ala Ile Lys Thr

545 550 555 560

Ala Val Glu Lys Leu Ala Thr Glu Ser Gln Ala Leu Gly Gln Ala Ile

565 570 575

Tyr Glu Ala Ser Ala Ala Asp Gln Ala Ala Gln Gly Asn Gly Ala Ala

580 585 590

Ala Pro Gln Ser Asp Asp Thr Val Val Asp Ala Glu Val Val Glu Glu

595 600 605

Pro Glu Lys Lys

610

<210> 42

<211> 126

<212> PRT

<213> Nocardiaceae

<400> 42

Met Ala Asn Val Asp Glu Leu Leu Glu Thr Phe Ala Gly Met Thr Leu

1 5 10 15

Leu Glu Leu Ser Glu Phe Val Lys Ala Phe Glu Asp Lys Phe Glu Val

20 25 30

Thr Ala Ala Ala Pro Val Ala Val Ala Val Ala Gly Gly Ala Ala Ala

35 40 45

Pro Ala Glu Ala Ala Glu Glu Gln Asp Glu Phe Asp Val Ile Leu Glu

50 55 60

Gly Ala Gly Asp Lys Lys Ile Gln Val Ile Lys Val Val Arg Glu Ile

65 70 75 80

Val Ser Gly Leu Gly Leu Lys Glu Ala Lys Asp Leu Val Glu Gly Ala

85 90 95

Pro Lys Pro Ile Leu Glu Lys Val Ala Lys Glu Ala Ala Glu Ala Ala

100 105 110

Lys Ala Lys Leu Glu Glu Ala Gly Ala Lys Ile Thr Val Lys

115 120 125

Claims

1. A preparation method of a DNA vaccine for fish nocardiosis comprises the following steps:

performing whole-bacterium protein electrophoresis on Nocardia seriolae, nocardia starchy and Nocardia salmonicida respectively by using a two-dimensional electrophoresis technology, comparing immunoblotting results of the three kinds of Nocardia seriolae, screening common immunoblotting protein points according to isoelectric points and molecular weights, wherein the common immunoblotting protein points are common antigens;

performing two-dimensional electrophoresis of the three nocardia whole proteins respectively, and cutting corresponding protein points on electrophoresis gel after dyeing by Coomassie brilliant blue according to the isoelectric points and molecular weights of common antigens to perform mass spectrometry and N-terminal sequencing;

identifying coding genes of the common antigen by combining mass spectrometry and protein sequencing;

selecting common antigen candidate genes after mutual verification according to the common antigen encoding genes and encoding genes of vaccine candidate common antigens predicted by reverse vaccinology;

designing primers according to sequences of common antigen candidate genes, wherein the forward primer and the reverse primer are respectively sequence No.1 and sequence No.2, or sequence No.3 and sequence No.4, or sequence No.5 and sequence No.6, or sequence No.7 and sequence No.8, or sequence No.9 and sequence No.10, or sequence No.11 and sequence No.12, or sequence No.13 and sequence No.14, cloning the corresponding primers into a vector to obtain the eukaryotic expression recombinant plasmid, the common antigen candidate genes are GroEL, sARp, dnaK, terd, FHA, rplL and PspA genes, the sequences of the GroEL, rpssA, dnaK, terd, FHA, rplL and PspA genes are respectively shown in sequence tables 29-35, the Nocardia seriolae is seriolae, nocardia asteroides, ATCC No. 0503, the Nocardia with the accession number of ZJ0503, the Nocardia with the accession number of ATCC 2747, and the Nocardia with the accession number of ATCC No. 19247.

2. The method of manufacturing according to claim 1, further comprising:

culturing engineering strain containing eukaryotic expression recombinant plasmid, and extracting recombinant plasmid without endotoxin in large amount.

3. Use of the process according to claim 1 or 2 for the preparation of a DNA vaccine against Nocardia disease in fish comprising Nocardia seriolae, nocardia stareri and Nocardia salmonicidae, wherein Nocardia seriolae is ZJ0503, nocardia stareri is deposited under the number ATCC 19247, and Nocardia salmonicidae is deposited under the number ATCC 27463.