CN108330143B - Edwardsiella tarda flagellin FliC with immune protection effect - Google Patents
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Abstract
The invention discloses an application of Edwardsiella tarda flagellin FliC with immune protection in subunit vaccine, which is prepared by a prokaryotic expression method and specifically comprises the following steps: amplifying the fliC gene by PCR; constructing a pMD18-T-fliC vector, and carrying out sequence determination on a fliC gene; carrying out amplification culture on the strain with the correct sequencing result, and extracting plasmids to obtain a purified target gene; constructing a recombinant expression vector pET-32 a-fliC; transforming the recombinant expression vector into DH5 alpha competent cells to obtain positive clones, extracting plasmids and sequencing; prokaryotic expression of the recombinant protein is carried out; analyzing the expression condition of the recombinant protein by SDS-PAGE; western blot verification is carried out on the recombinant protein. According to the invention, a large amount of purified recombinant protein of the Edwardsiella tarda flagellin FliC is obtained by a prokaryotic expression method, and the immunogenicity and the protection of a model animal of the recombinant protein are further evaluated by using an animal experiment, which indicates that the Edwardsiella tarda flagellin FliC can be used for developing subunit vaccines.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to edwardsiella tarda flagellin FliC with immune protection effect.
Background
Edwardsiella tarda (Edwardsiella tarda, E.tarda) is seriously harmed on aquaculture, the death rate of fishes infected with the Edwardsiella tarda is high, the fishes are easy to repeat, the immunity is low, the curative effects of traditional antibiotics and chemical medicines are not exact, and a novel, efficient and safe prevention and treatment method is urgently needed to be developed. The great success of the vaccine in livestock and poultry production provides a great reference for aquaculture, a plurality of aquatic vaccines are developed and applied, good prospects are shown, and unfortunately, no commercial vaccine is used for preventing and controlling the infection of Edwardsiella tarda at present. In addition, Edwardsiella tarda can cause various infections of people, and in recent years, reports of infections of people through food sources are increasing, and the public health value of the Edwardsiella tarda is revealed.
Flagellin is one of the main structural proteins of bacterial flagella, plays an important role in the processes of bacterial movement, adhesion and host infection, and is an important virulence factor of bacteria. The research finds that the bacterial flagellin can activate host immune response and act as vaccine adjuvant, and the further research finds that the flagellin can induce inflammatory response to occur and promote dendritic cell maturation through TLR5 channel. The protein shows good immune effect no matter mixed with antigen or fusion expression. In addition, the flagellin has strong immunogenicity and certain immune protection. FliC is a major protein component constituting flagella filaments, is a focal protein of current flagella research, and has been analyzed to some extent for biological functions. According to the invention, a large amount of purified recombinant protein of the Edwardsiella tarda flagellin FliC is obtained by a prokaryotic expression method, and the immunogenicity and the protection of a model animal of the recombinant protein are further evaluated by using an animal experiment, which indicates that the Edwardsiella tarda flagellin FliC can be used for developing subunit vaccines.
Disclosure of Invention
The invention aims to provide Edwardsiella tarda flagellin FliC with immune protection effect, and particularly obtains Edwardsiella tarda flagellin FliC recombinant protein by a prokaryotic expression method, thereby further developing an Edwardsiella tarda subunit vaccine.
In order to achieve the purpose, the invention adopts the following technical scheme:
the application of Edwardsiella tarda flagellin FliC with immune protection function in subunit vaccine.
Further, the preparation method of the edwardsiella tarda flagellin FliC with the immune protection effect comprises the following specific steps:
(1) performing PCR amplification on the fliC gene by taking an Edwardsiella tarda ET-CL genome sequence as a template and P1 and P2 as primers; the reaction system for PCR amplification was 20. mu.L, 2 XTaq PCR Mix 10. mu.L, template 1. mu.L, primers P1 (20. mu.M) and P2 (20. mu.M), each 1. mu.L, ddH2O7 mu L; the reaction program of PCR amplification is pre-denaturation at 94 ℃ for 5min, at 94 ℃ for 30s, at 54 ℃ for 30s, at 72 ℃ for 60s, for 25 cycles, at 72 ℃ for 10 min; wherein the primers P1 and P2 are:
P1：5’-ATGGCACAAGTAATTAATACC-3’；SEQ ID NO：1；
P2：5’-ACGCAGCAGAGACAGGACG-3’；SEQ ID NO：2；
(2) constructing a pMD18-T-fliC vector, and carrying out sequence determination on a fliC gene;
(3) carrying out amplification culture on the strain with the correct sequencing result, extracting a plasmid, carrying out PCR amplification on the fliC gene by using pMD18-T-fliC as a template and P3 and P4 as primers to obtain a purified target gene; the reaction system for PCR amplification was 20. mu.L, 2 XTAQPCR Mix 10. mu.L, primers P3 (20. mu.M) and P4 (20. mu.M) each 1. mu.L, template 1. mu.L, ddH2O7 mu L; the reaction program of PCR amplification is pre-denaturation at 94 ℃ for 5min, at 94 ℃ for 30s, at 52 ℃ for 30s, at 72 ℃ for 60s, for 30 cycles, at 72 ℃ for 10 min; it is composed ofThe middle primers P3 and P4 are:
P3：5’-CGGGATCCATGGCACAAGTAATTAATACC-3’，BamHⅠ；SEQ ID NO：3；
P4：5’-GCGTCGACACGCAGCAGAGACAGGACG-3’，SalⅠ；SEQ ID NO：4；
wherein the underlined part is the restriction site;
(4) carrying out double enzyme digestion on the purified target gene and the pET-32a vector by utilizing restriction enzymes BamH I and Sal I respectively to construct a recombinant expression vector pET-32 a-fliC;
(5) transforming the recombinant expression vector into DH5 alpha competent cells, selecting a single clone to perform PCR and double enzyme digestion verification to obtain a positive clone, extracting a plasmid, and sequencing;
(6) the recombinant expression vector pET-32a-fliC and the empty vector pET-32a are respectively transformed into BL21 engineering bacteria for prokaryotic expression of recombinant protein, and the specific steps are as follows: respectively adding 5 mu L of recombinant vector pET-32a-fliC and 5 mu L of empty vector pET-32a into 100 mu L of BL21 engineering bacteria; performing ice bath for 30min, performing heat shock at 42 ℃ for 90s, and performing ice bath for 5 min; adding 1mL of liquid LB culture medium, rejuvenating at 37 ℃ for 1h, respectively taking 5 mu L of solid LB plates coated with 100 mu g/mL final concentration of ampicillin, inverting, and culturing at 37 ℃ overnight;
(7) analyzing the expression condition of the recombinant protein by SDS-PAGE;
(8) and carrying out Westernblot verification on the recombinant protein by using His-Mab as a primary antibody and HRP-IgG as a secondary antibody.
The invention has the beneficial effects that: according to the invention, a large amount of purified recombinant protein of the Edwardsiella tarda flagellin FliC is obtained by using a prokaryotic expression method, and the immunogenicity and the protection of the recombinant protein on model animals are further evaluated by using animal experiments, so that the subunit vaccine and the nucleic acid vaccine target of the Edwardsiella tarda are further developed, and meanwhile, the Edwardsiella tarda FliC protein also has adjuvant properties.
Description of the drawings:
FIG. 1 shows PCR amplification of fliC gene in example 2 of the present invention;
m.dl2000marker; fliC gene amplification;
FIG. 2 is a SDS-PAGE analysis of the expression of recombinant proteins in E.coli in example 4 of the present invention;
m. protein Marker; 1, 2.pET-32a-fliC/BL21 thalli lysate; pET-32a/BL21 empty control;
FIG. 3 shows the expression of the Westernblot identified recombinant protein in E.coli in example 4 of the present invention;
1. a protein Marker; pET-32a-fliC/BL21 thalli lysate;
FIG. 4 is a solubility analysis of the recombinant protein in example 5 of the present invention;
m. protein Marker; pET-32a-fliC/BL21 thalli lysate; 2. a lysate supernatant; 3. precipitating a lysate;
FIG. 5 is an SDS-PAGE of the target protein purified in example 5 of the present invention;
m. protein Marker; 1. unpurified recombinant protein; 2. purifying the recombinant protein;
FIG. 6 shows the His-FliC protein recognition by Westernblot analysis positive sera in example 6 of the present invention;
1. a recombinant protein; 2. negative control (His- σ C reovirus capsid protein);
FIG. 7 shows the immunoprotective effect of His-FliC protein in example 6 of the present invention on mice;
FIG. 8 is a graph showing the monitoring of BSA antibody titers in the different adjuvants of example 7 of the present invention;
FIG. 9 shows anti-FliC antibody titer monitoring after His-FliC immunized mice in example 7 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the attached drawings, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention.
Example 1 sequence analysis of fliC Gene of Edwardsiella tarda
Primers were designed based on the e.tarda reference strain ET-1 genomic sequence (CP001135.1) fliC gene registered on GenBank, primers P1 and P2 were:
P1：5’-ATGGCACAAGTAATTAATACC-3’；SEQ ID NO：1；
P2：5’-ACGCAGCAGAGACAGGACG-3’；SEQ ID NO：2；
extracting an ET-CL genome by using an OMEGA genome extraction kit, and performing PCR amplification by using the ET-CL genome as a template; the reaction system for PCR amplification is 2 XTaqPCR Mix 10. mu.L, primers P1 (20. mu.M) and P2 (20. mu.M) each 1. mu.L, template 1. mu.L, ddH2O7 mu L, 20 mu L in total; the reaction program of PCR amplification is pre-denaturation at 94 ℃ for 5min, at 94 ℃ for 30s, at 54 ℃ for 30s, at 72 ℃ for 60s, for 25 cycles, at 72 ℃ for 10 min; recovering the fliC gene segment, connecting with a PMD18-T vector by the following connection system: mu.L of the target gene, 0.5. mu.L of pMD18-T Vector and 5. mu.L of 2 XConnectbuffer, 10. mu.L in total, and ligated at 16 ℃ overnight. The ligation product was transformed into DH5 α competent cells; single colony is picked for PCR detection, and positive bacteria are sent to Beijing for sequencing.
The fliC gene conservation was analyzed with reference to the fliC gene sequence of the e.tarda reference strain (e.tarda et-1, e.tarda ETW41, e.tarda FL6-60) and other members of the enterobacteriaceae family (s.marcocens str.smunaam836, Klebsiella aerogenes str.ar _0009, s.typhimurium str.lt2, e.coli str.k-12, s.enterica str.sgb23, Kosakonia cowanister.esp _ Z) strains in GenBank, and as a result, the fliC gene was found to be highly conserved among the edwardsiella members.
Example 2 Edwardsiella tarda fliC Gene amplification
The PMD18-T-fliC strain with correct sequencing result in example 1 is subjected to amplification culture, plasmids are extracted, and the plasmid is used as a template, P3 and P4 are used as primers, the fliC gene is amplified by PCR, the total reaction system is 20 mu L, wherein 2 xTaq PCR Mix is 10 mu L, the primers P3(20 mu M) and P4(20 mu M) are 1 mu L respectively, the template is 1 mu L, and ddH is 1 mu L2O7 mu L; the reaction program is pre-denaturation at 94 ℃ for 5min, 30s at 94 ℃, 30s at 52 ℃, 60s at 72 ℃ for 30 cycles, and 10min at 72 ℃; obtaining a target fragment (shown in figure 1) with the size of 1248bp, purifying a target gene, and connecting the target gene with a prokaryotic expression vector pET-32a to construct a recombinant expression vector; wherein the primers P3 and P4 are:
P3：5’-CGGGATCCATGGCACAAGTAATTAATACC-3’，BamHⅠ；SEQ ID NO：3；
P4：5’-GCGTCGACACGCAGCAGAGACAGGACG-3’，SalⅠ；SEQ ID NO：4；
in which the underlined part represents the cleavage site.
EXAMPLE 3 construction of pET-32a-fliC recombinant expression vector
And (3) carrying out double enzyme digestion on the purified target gene and the pET-32a vector by using BamHI and SalI respectively, wherein the enzyme digestion system comprises the following steps: the target gene/pET-32 a was 25. mu.L, BamHI and SalI were each 1. mu.L, and 10 XGreenbuffer was 3. mu.L. After recovery, the ligation products were ligated at 22 ℃ for 30min in a ratio of 3:1, all the ligation products were transferred to 100. mu. LDH 5. alpha. competent cells, ice-bathed for 30min, heat bathed for 90s at 42 ℃ and ice-bathed for 5min, 1mL LB liquid medium was added, rejuvenated at 37 ℃ for 1h, spread on a solid LB plate containing 100. mu.g/mL final concentration of ampicillin, and cultured overnight at 37 ℃ by inverting. The next day, single clones were picked for PCR validation using primers P3, P4. And (3) carrying out double enzyme digestion verification and sequence determination on the positive clone extracted plasmid, and obtaining a recombinant expression vector pET-32a-fliC without mutation and frame shift.
EXAMPLE 4 prokaryotic expression of recombinant proteins
Respectively transforming the recombinant vector pET-32a-fliC and the empty vector into BL21 engineering bacteria, carrying out ice bath for 30min, carrying out heat bath for 90s at 42 ℃ and carrying out ice bath for 5 min; 1mL of LB liquid medium was added, rejuvenated at 37 ℃ for 1 hour, spread on a plate containing 100. mu.g/mL of ampicillin at a final concentration, and incubated overnight at 37 ℃. A single colony is selected and inoculated in a liquid LB culture medium containing 100 mu g/L ampicillin, shake culture is carried out at 37 ℃ until logarithmic phase, IPTG is added until the final concentration is 0.5mmol/L, and induced expression is carried out for 5 h. Centrifugally collecting thalli precipitates, and washing for 3 times by using PBS (phosphate buffer solution); adding protein sample buffer solution, mixing, boiling for 10min, centrifuging, collecting supernatant as protein sample, analyzing with SDS-PAGE, loading, adjusting voltage to 80V, separating the sample with separating gel and concentrated gel, adjusting voltage to 120V, collecting the sample, performing electrophoresis, dyeing with Coomassie brilliant blue for 1min, and boiling with distilled water for several times for decolorizing; as shown in FIG. 2, the desired protein was expressed at 60 kD; His-MAb is used as a primary antibody, HRP-IgG is used as a secondary antibody, 5% skimmed milk powder is respectively diluted according to the ratio of 1:1000 and 1:5000, and the target protein expression is further confirmed by a Westernblot method, which comprises the following specific steps: after SDS-PAG electrophoresis, taking out the film, transferring the protein to an NC film by using a film transfer instrument, and transferring the protein to the NC film for 1h at 80V; after the membrane transfer is finished, taking out the NC membrane, and washing the NC membrane for 3 times and 5 min/time by PBST; sealing 5% skimmed milk powder at room temperature for 1h, washing with PBST for 3 times, 5 min/time; diluting His-Mab with 5% skimmed milk powder at a ratio of 1:1000 as primary antibody, and incubating at room temperature for 1 h; PBST washing 3 times, 5 min/time; diluting HRP-IgG as a secondary antibody by 5 percent of skimmed milk powder according to a ratio of 1:5000, and incubating for 30min at room temperature; PBST washing 3 times, 5 min/time; adding developing solution, keeping out of the sun for 5min, and finally performing luminescence development; as a result, as shown in FIG. 3, a band of the objective protein appeared at 60kD, which confirmed successful expression of the His-FliC recombinant protein.
Example 5 solubility analysis and purification of the protein of mesh
Carrying out amplification culture on the strain expressing the target protein according to the conditions, and centrifugally collecting thalli; the bacterial cells were disrupted by ultrasonication, and the precipitates were separated by centrifugation and the supernatant, and the bacterial lysate, the supernatant and the precipitate were each prepared into protein samples, and SDS-PAGE was carried out, whereby protein bands appeared at 60kD, as shown in FIG. 4.
Taking the induction expression supernatant, and using Ni2+As a result of purification by affinity chromatography, His-FliC was purified at 60kD, and as a result of determination of the purified protein concentration by the BCA protein concentration detection kit, the protein concentration was 0.36mg/mL, as shown in FIG. 5.
Example 6 recombinant FliC protein immunoprotection experiments
The positive serum of the E.tarda-infected mouse is prepared, the positive serum (1: 500) is used as a primary antibody, HRP-IgG (1:5000) is used as a secondary antibody, the recognition of the positive serum to the recombinant protein is verified by Western blot, and the result is shown in figure 6.
40 healthy Kunming mice were randomly divided into two groups, a control group and an immunized group, with 20 mice per group. Using purified His-FliC protein as immunogen, immunizing mice in an immunization group for the first time, injecting 30 mu g of recombinant FliC protein (Freund's complete adjuvant emulsification) into muscles, carrying out the second immunization (Freund's incomplete adjuvant emulsification) after 2 weeks, and strengthening the immunization by the Freund's incomplete adjuvant emulsification protein after 30 days; control mice were immunized three times with an equal volume of PBS (plus corresponding adjuvant). 48 th day, the two groups of mice are inoculated with Edwardsiella tarda ET-CL bacterial liquid in the abdominal cavity, and the dosage is 5LD50(2.0×107cfu), 4d post-infection, and statistics of mortalityAmount of the compound (A). The results showed that all the mice in the control group died, 6 mice in the immune group died, and the rest mice gradually recovered to health after experiencing transient mental depression and no food. The results are shown in fig. 7, which indicates that the His-FliC recombinant protein has certain protection power after immunizing mice, and the protection rate is 70%.
Example 7 evaluation of the Effect of Edwardsiella tarda FliC protein as an adjuvant
40 healthy Kunming mice were randomly divided into 4 groups of 10 mice each. The purchased bovine serum albumin BSA was used as an evaluation protein, and a BSA immunization group, a BSA + FliC protein immunization group, and a BSA + Freund's adjuvant immunization group were set, and a PBS group was also set as a control. The immunization route is subcutaneous injection, the immunization is carried out in two times, the second immunization is carried out at an interval of 10 days after the first immunization, and the protein immunization dose is 100 mu g/mouse. The BSA protein oil emulsion immunization group is immunized by adding complete Freund adjuvant in the first immunization and adding incomplete Freund adjuvant in the second immunization, and the blank control group is replaced by PBS.
Collecting blood from tail tip of each immunization group at 0, 10, 20 and 30 days after immunization, separating serum, detecting antibody titer in mouse serum by indirect ELISA method with purified recombinant FliC protein as coating antigen, and using negative serum OD450Adding 3 times of standard deviation to the average value to serve as a negative and positive judgment standard, calculating a critical value to be 0.176, and judging the serum to be detected to be positive when the OD value is larger than the critical value; and when the OD value of the serum to be detected is less than the critical value, judging the serum to be negative.
Serum collected from 4 times of mice immunized with different adjuvants and BSA was diluted at a ratio of 1:100, 500ng/ml BSA protein was used to coat the ELISA plate, and goat anti-mouse IgG-HRP was used as a secondary antibody for indirect ELISA detection, with the results shown in FIG. 8. The results show that: the mouse immunized by pure BSA hardly produces specific antibodies, and Freund's adjuvant or FliC mixed immunization can stimulate the organism to produce higher specific antibodies, thus proving the adjuvant property of the Edwardsiella tarda FliC protein.
Serum collected from 4 FliC immunized mice was diluted 1:100, 500ng/ml recombinant protein was used to coat the ELISA plate, and goat anti-mouse IgG-HRP was used as a secondary antibody for indirect ELISA detection, with the results shown in FIG. 9. The results show that: the recombinant protein immunized mice can produce high-level specific antibodies, and the highest level is achieved after the boosting immunization.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
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Claims (4)
1. The application of Edwardsiella tarda flagellin FliC with immune protection in preparing subunit vaccine is characterized in that the preparation method of the subunit vaccine is as follows:
(1) performing PCR amplification by taking an Edwardsiella tarda ET-1 genome sequence as a template and P1 and P2 as primersfliCA gene; wherein the primers P1 and P2 are:
P1：5’-ATGGCACAAGTAATTAATACC-3’ SEQ ID NO：1；
P2：5’-ACGCAGCAGAGACAGGACG-3’ SEQ ID NO：2；
(2) construction of pMD18-T-fliCCarrier, pairfliCSequencing the gene;
(3) amplifying and culturing the strain with correct sequencing result, extracting the plasmid, and performing pMD18-T-fliCAs template, P3 and P4 as primers, PCR amplificationfliCGene, obtaining purified target gene; wherein the primers P3 and P4 are:
P3：5’-CGGGATCCATGGCACAAGTAATTAATACC-3’，BamHⅠ；SEQ ID NO：3；
P4：5’-GCGTCGACACGCAGCAGAGACAGGACG-3’，SalⅠ；SEQ ID NO：4；
(4) using restriction endonucleasesBamHI andSali, respectively carrying out double enzyme digestion on the purified target gene and the pET-32a vector to construct a recombinant expression vector pET-32a-fliC；
(5) Transforming the recombinant expression vector into DH5 alpha competent cells, selecting a single clone to perform PCR and double enzyme digestion verification to obtain a positive clone, extracting a plasmid, and sequencing;
(6) recombinant expression vector pET-32a-fliCAnd the empty vector pET-32a are respectively transformed into BL21 engineering bacteria to carry out prokaryotic expression of recombinant protein;
(7) analyzing the expression condition of the recombinant protein by SDS-PAGE;
(8) carrying out Western blot verification on the recombinant protein;
(9) taking the induction expression supernatant, and using Ni2+Purifying by affinity chromatography to obtainProtein FliC;
(10) emulsifying the protein FliC obtained in the step (9) by using a Freund complete adjuvant to obtain a subunit vaccine;
the specific steps for converting the BL21 engineering bacteria in the step (6) are as follows: respectively adding 5 μ L of recombinant vector pET-32a-fliCAnd 5 mul of empty vector pET-32a are added into 100 mul of BL21 engineering bacteria; performing ice bath for 30min, performing heat shock at 42 ℃ for 90s, and performing ice bath for 5 min; after adding 1mL of liquid LB medium and rejuvenating at 37 ℃ for 1 hour, 5. mu.L of each was spread on solid LB plates with a final concentration of ampicillin of 100. mu.g/mL, inverted and incubated overnight at 37 ℃.
2. The use of the Edwardsiella tarda flagellin FliC with immune protection effect in preparing subunit vaccine according to claim 1, characterized in that the reaction system of the PCR amplification in step (1) is 20 μ L in total, wherein 2 xTaq PCR Mix is 10 μ L, 20 μ M P11 μ L, 20 μ M P21 μ L, template 1 μ L, ddH2O7 mu L; the reaction procedure of PCR amplification is pre-denaturation at 94 ℃ for 5min, at 94 ℃ for 30s, at 54 ℃ for 30s, at 72 ℃ for 60s, for 25 cycles, and at 72 ℃ for 10 min.
3. The use of the Edwardsiella tarda flagellin FliC with immune protection effect in preparing subunit vaccine according to claim 1, characterized in that the reaction system of the PCR amplification in step (3) is 20 μ L in total, wherein 2 xTaq PCR Mix is 10 μ L, 20 μ M P31 μ L, 20 μ M P41 μ L, template 1 μ L, ddH2O7 mu L; the reaction procedure of PCR amplification is pre-denaturation at 94 ℃ for 5min, at 94 ℃ for 30s, at 52 ℃ for 30s, at 72 ℃ for 60s, for 30 cycles, and at 72 ℃ for 10 min.
4. The use of the edwardsiella tarda flagellin FliC with immune protection effect in preparing subunit vaccine according to claim 1, wherein the Western blot in step (8) verifies His-Mab as primary antibody and HRP-IgG as secondary antibody.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105920593A (en) * | 2016-06-08 | 2016-09-07 | 山东省海洋生物研究院 | Edwardsiella tarda subunit oral microencapsule vaccine for aquatic product |
| CN106075418A (en) * | 2016-06-08 | 2016-11-09 | 类延乐 | A kind of fliC albumen and GAPDH albumen microcapsule vaccine |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105920593A (en) * | 2016-06-08 | 2016-09-07 | 山东省海洋生物研究院 | Edwardsiella tarda subunit oral microencapsule vaccine for aquatic product |
| CN106075418A (en) * | 2016-06-08 | 2016-11-09 | 类延乐 | A kind of fliC albumen and GAPDH albumen microcapsule vaccine |
Non-Patent Citations (6)
| Title |
|---|
| Base changes in the fliC gene of Edwardsiella tarda: possible effects on flagellation and motility;Jun Okuda et al;《DISEASES OF AQUATIC ORGANISMS》;20090630;第76卷;第113-121页 * |
| Construction and evaluation of DNA vaccines encoding Edwardsiella tarda antigens;Xu-dong Jiao et al;《Vaccine》;20090709;第5195-5202页 摘要，材料与方法，结果，讨论部分 * |
| Edwardsiella tarda flagellar protein FlgD：A protective immunogen against edwardsiellosis;MengZhang et al;《Vaccine》;20120420;第30卷;第3849-3855页 摘要，第2-4节 * |
| FliC, a Flagellin Protein, Is Essential for the Growth and Virulence of Fish Pathogen Edwardsiella tarda;Yang He et al;《PLOS ONE》;20120930;第7卷(第9期);e45070 * |
| 迟缓爱德华氏菌（Edwardsiella tarda）的多样性分析及其鞭毛蛋白的功能研究;贺扬;《中国博士学位论文全文数据库 农业科技辑》;20120215(第02期);D052-21 * |
| 迟缓爱德华氏菌基因工程疫苗的构建及免疫效果的研究;焦绪栋;《中国博士学位论文全文数据库 医药卫生科技辑》;20101015(第10期);摘要，第2-5章 * |
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