LU501952B1 - Yeast engineering bacteria for preventing porcine circovirus disease - Google Patents

Abstract

This invention provides yeast engineering bacteria for preventing porcine circovirus disease. The gene engineering bacteria contain the gene encoding the LSO fusion protein, and the sequence of the LSO fusion protein is shown in SEQ-ID-NO:1. This invention uses the surface of the yeast integrated plasmid with high copy to display both the multi-epitope antigen of porcine circovirus PCV2 and the fusion protein of protective antigen LTB-SLT-IIeB of pathogenic porcine colibacillosis. The recombinant fusion protein LTB-SLT-IIeB itself has the function of immune adjuvant, that is, as an antigen and adjuvant, and it plays a dual role; and it improves that immunogenicity and immune efficacy of recombinant beer yeast strain to display the CAP protein encoded by ORF2 of porcine circovirus PCV2. The yeast engineering bacteria of the invention can be applied to the prevention and treatment of porcine circovirus disease.

Description

Description 10501952 Yeast engineering bacteria for preventing porcine circovirus disease

Technical Neid

The invention belongs to the technical field of genetic engineering, particularly relates to yeast enginsering bacteria for preventing porcine circovirus disease,

Background

Porcinecircovirustypez (PCV-2) is the main pathogen of post weaning mulli-systemic wasting syndrom (PMVS} Since the first outbreak of the disease in Canadian pigs in 1991, it has been widely spread among pigs all over the world, causing huge economic losses to the world pig industry.

PCY-Z is often associated with porcine reproductive and respiratory syndrome virus {(PRRSVYY, porcineparvovirus, PPY), porcine pseudoratiesvirus (PRY), Haemophilusparasuis(HPsgy, Actinonacillus pleuropneumonias (APP), swine Pasteurellosis {SP} and many other pathogens have concurrent or secondary infection, which increases the difficulty of prevention and control, Besides PMWS, FCV-2 can also cause dermatitis and nephrotic syndrome (PDNS) in fattening pigs, porcine respiratory syndrome (PROC), profiferative necrotizing prneumonia (PNP) and diarrnea in newbom piglets.

Besides PRWS, PCV-2 can also cause dermatitis and nephrotic syndrome (PONS) in growing and fattening pigs, porcine respiratory disease complex (PRDC) proliferative necrotising pneumonia (PNP) and diarrhea in newborn piglets, According to incomplete estimates, COTCINE CICOVITUS associated disease(PCVAD) caused economic losses of more than 600 million euros in Europe in 2005. POVAD is also one of the important threats to Ching’s pig industry, and à has also caused serious economic losses to Chinas pig production At present, there are many unsoived problems in the epidermiclogicai characteristics of porcine circovirus type 2, the pathogenic mechanism of the virus, and the research on prevention and control of the virus itself and PCVAD.

Moreover, there are still many controversies about the diagnostic criteria, pathogenic pathogens and prevention and control measures of PCVAD, Although vaccines against PCV-2 are on the market at home and abroad, there are still some problems to be solved, including the reliability of clinical immune affect and the high cost of vaccines.

In recent years, it has been found that although the number of syndrome cases caused by PCOV-2 has decreasad worldwide, the gpidemiciogical survey results of domestic scholars show that the infection rate of POV 00. pigs is on the rise, which is tantamount to burying a time bomb in pigs, which may endanger the health of pigs at any time and cause serious sconomic losses. PCV-2 has been devastating the pig flock for more than 20 years. No matter ai the research level or the production level, it is required Io make a big bragkihrough in the detection and prevention of PCY-Z infection, in order to protect the pig production.

The highly affective PCV-2 vaccine is one of the research focuses of scholars at home and abroad. Since 2006, PCY-2 vaccines have been put on the market in the United Sales and Europe, which are mainly inactivated vaccines and subunit vaccines, PCY-2 inactivated vaccine has the advantages of non-foxicity, safety and stable performance. Inactivated POV-2 vaccine containing adiuvant has been applied commercially and licensed in some European countries. Circovac, a whole virus inactivated vaccine developed by Merial Company, is one of the representatives.

Suvaxyn PCV-Zonedose of FortDodge Company has been licensed for clinical application in the United States, If is mainly used for immunization of piglets, which can sfiectively prevent POV-2 toxemia, reduce the damage of lymphalic tissue and the mortality of piglets, and the immune response stimulated is not affected by maternal antibodies (Xing Hatyun et al, 2009) Some domestic scientific research institutes have developed POV-2 inactivated vaccins, and the results show thai the vaccine can shorten the time of viral hematopathy and reduce the virus-carrying capacity of pigs. However, the inactivated vaccine of PCV-2 has some disadvantages, such as short stimulation time to organism, heavy Siress 10 pigs, repeated inoculation with unsatisfactory effect, and low proliferation titer of PCV-2 on cells, so its clinical value is ow.

The nucleocapsid protein Cap of POV-2 is the main immunogenic protein of the virus, and has no cross-protective effect with PCV-1Cap protein. Therefore, researchers at home and abroad expressed Cap protein separately in different expression systems to develop subunit vaccines which can effectively prevent and treat POV-2 infection, At present, subunit vaccines have been applied clinically, including Forcils PCY developed by AKZO-NOBEL and CirooFLEX developed by Boehringer Ingelheim Company. Both these subunit vaccines are the expression products of baculovirus expression system, which has the advantage of expressing protein in baculovings 99 expression system, and has good immune effect (Xinge et ai, 2009).

Blanchard ef al (Blanchardeta 1., 2006) compared the immune efficacy of POV-2 gens vaccins and subunit vaccins, and found that the effect of two immunizations with PCV-2 gene Vaccine alone was lower than that of subunit vaccine, it can't completely inhibit the proliferation of FCV-2, Compared with other vaccines, subunit vaccins produces antibodies earlier and the antibody level is more stable. it can effectively prevent the replication and pathological influence of PCV-2 In vivo, and it is safer and more convenient io use. Therefore, the subunit vaccine of Cap protein has a wide application prospect in the prevention and treatment of POV-2 related diseases, and its clinical immune effect has also Leen affirmed. At present, the existing problem is that is production cost is high, which is not conducive IC large-scale immunization.

Af present, commercial vaccins has become a relatively successful means to control FOYE, and the immune effect of subunit vaccine is better than that of whole virus inactivated vaccine and the cost is relatively low.

From the pathogenesis of circovirus itself and the mechanism of immune response {the immune response of circovirus takes a long time to produce neutralizing antibody), one shot of inactivated whole virus vaccine or subunit vaccine will not produce ideal effect, while the single shot of commercial vaccine costs 9 ~ 20 RMB for domestic vaccine and 29 ~ 58 yuan Renminbi for imported vaccine, Moreover, the potential stress cost of two or more intramuscular injections In pigs Is also astonishing. Under the premise of ever increasing bresding cost, in view of the defects of the technology itself and its unusualiy high frnunization cost (even the cost has exceeded the normal profit of pig breading), this has become the bottleneck of the current commercial vaccine immunization control PCOV-2.

Therefore, it is the focus of current research to find products with low cost that can fully induce humoral immunity and cellular immunity. Moreover, the way of using the product should best reflect the direction of environment-friendiy" pig raising.

Yeast display technology is a new cell surface display technology developed after phage display technology. The basic principle is that o-lectin and o-lectin related io yeast matching type are used as skeleton proteins to fuse with protein or polypeptides encoded by foreign genes, so that protein or polypeptide encoded by foreign genes can be expressed 99 on ihe surface of veast cell wall, This technique has been successfully applied to molecular direcled evolution of protein, affinity maturation and identification of antibodies, and epitopes analysis of protein. Because yeast is a safe organism, i is widely used in pharmaceutical industry and food industry. For example, brewer's yeast has been used orally io rest dysentery caused by human intractable Clostridium; In vivo experiments in mice show that Saccharomyces cerevisiae has the effect of anti-influenza virus.

Yeast has many advantages as the carrier of live vaccine. The recombinant foreign protein is expressed in the form of secretion, and iis folding form is closer to the orientation of natural protein, so its immunogenicity is closer to that of natural protein. The recombinant axogenous antigen molecules have high expression level, and 105 antigen molecules can be expressed on the surface of each veast cell These molecules can promote the interaction between yeast cells and immune cells in the form of multivalent cross-ligating and start the signal transmission of immune cells, Antigen molecules are expressed on the surface of yeast cells, which are easier to be recognized by immune cells, Even small peptides show immunogenicity on the surface of yeast cells, Foreign microbial cell surface display technology has been used to construct oral live vaccine, and the most successful example is the meliitin Ag2.5 displayed on the surface of streptococcus settled in human oral cavity, Using this recombinant bacteria once orally or intranasally to immunize mice, the recombinant bacteria can stay in the oral and nasal mucosa of mice for 10-11 weeks, and high titers of secretory IgA against Ag25 can be detected in tha saliva and lungs of mice, at the same time, specific 1506 against Ag2.5 can also be detected in the serum of mice.

in addition, human hepatitis B surface antigen (HBsAg) displayed by beer yeast can stimulate mice to produce anti-HbsAg specific 196. It shows that non-pathogenic microorganisms arg used as vaccine carriere, and specific antigenic determinants are expressed on the surface of microorganisms. | is feasible to construct a new oral live vaccine, but its shortcomings need to be further improved. In China, the research on using rriicrobial surface display technology to construct oral vaccins started late, and there is no formal report yet.

At present, the main problems of vaccine construction by microbial surface display 95 technology are that the recombinant strain stays on the mucosa for a short time, and the expression level of foreign protein is too low to effectively stimulate mucosal immunity.

Summary This invention provides yeast engineering bacteria for preventing porcine circovirus disease.

This invention provides a technical solutions as followings: À genetically engineered baclerium contains the gene encoding the LSO fusion protein, and the sequence of the LSO fusion protein is shown in SEQUID NO: 1 Praferably, the gene encoding LSO fusion protein is obtained by modifying LTE gene, SLT-HeB gene in pathogenic Escherichia coli and ORF2 gene of encoding viral ruiclencapsid protein Cap according to codon preference of genstic engineering bacteria, and it is obtained by ligating with linker sequence.

Preferably, the engineering bacteria is besr yeast.

Preferably, the nucleic acid sequence of encoding the LSO fusion protein is shown in SEOIDNO 2.

Freferabiy, the LSO gene was transferred into the vector pHEMSGSAC to obtain the expression plasmid pHBMSGSAC-LSO.

Preferably, the expression plasmid pHBMSGSAC-LSO was transferred into Saccharomyces cerevisiae Io obtain yeast genetic engineering bacteria Preferably, the yeast is Saccharomyces cerevisiae INVSCL.

Application of the genetically enginesred bacteria of claim 1 in prevention and treatment of porcing CICOVITUS disease.

Application of the genstic engineering bacteria of claim 1 in preparing genetic engineering vaccine of porcine circovirus disease, Prefsrably, the construction method of the yeast engineering bacteria comprises the following steps: 1) Modifying the LTB gene, SLT-HNeBß gene and ORFZ of porcine circovirus type 2 coding virus nucleocapsid protein Cap in pathogenic Escherichia coli according fo veast codon preference;

2) Using overlapping PCR technology to amplify the modified LTB, SLT-lleB Baha 9 ORFZ, and the full length of LEO fusion gene is obtained by connecting the connecting sequence, 3} Saccharomyces cerevisiss display expression vector pHEMSGSAC is treated by Cpo ! and Not | double digestion 4} Two nucleotide sequences of "GTAC” and "GGCCA" are introduced info the two ends of the fusion gene L300, and in the presence of dTTP, the PCR product is treated with T4DNA polymerase to obtain the sticky end; 5) inserting the fusion gene LSO into the vector pHEM3GEAC by DNAligase to form the recombinant plasmid pHEMS3GSAC-LS3O; GS) Plasmid pHBM36BAC-LSO is linearized by Hpal digestion, the resistance gene and replication point of E.coli are cut off, and then is transfected into competent calls of Saccharomyces ceravisias INVSCL by LiAc method, next, coating with auxotrophic culture medium, and the positive recombinant strain INVSCIOMEMSSSAC-LSO was screensd: 7) Using recombinant bacteria as template, the target gene was amplified by FOR specific primers, in order to further verify the positive recombinant bacteria.

The beneficial effects of this invention are described as followings: This invention uses the surface of the yeast integrated plasmid with high copy to display both the multi-epitope antigen of porcine circovirus PCV2 and the fusion protein of protective antigen LTB-SLT-lleB of pathogenic porcine colibaciliosis. The recombinant fusion protein LTB-SLT-lleB itself has the function of immune adjuvant, thal is, as an antigen and adjuvant, and it plays a dual role, moreover, the engineering yeast strain retains the characteristics of the parent stran INVSCL as an immune adjuvant of beer yeast, which improves immunogenicity and immune efficacy of recombinant beer yeast strain to display the CAP protein encoded by ORF2 of porcine circovirus POV2. The yeast engineering bacteria of the invention can be applied to the prevention and treatment of DOrCINS circovirus disease. The integrated multi-copy tandem repeated expression strategy was used to solve the problems of low expression level and unstable expression.

The immunogenic gene in the genetic engineering strain of the invention is displayed and expressed on the surface of beer yeast The recombinant strain does not contain other gxoganous genes except LSO gens, and retains the characteristics of the parent strain

INVSCL as an immune adjuvant of beer veast The recombinant strain can be directly sed 998 as a vaccine after freeze-drying process without extracting protein. In this way, the immune adjuvant effect of beer yeast can be exerted, which can not only improve the nonspecific immunity of inoculated pigs, but also improves the spacific immunity of pigs.

The genetic engineering strain of the invention can be fermented at high density, with the ODSC00 above 40, without adding inducers in the whole fermentation process, and can be expressed at the end of lnganthmic phase strictly with high efficiency.

The genetic engineering strain prepared by the invention does not contain resistance markers; in addition, Saccharomyces cerevisiae IS a food safety microorganism, which is widely used in food production, and does not have the problem of infecting the host, thus completely meeting the requirements of vaccine biosafety.

Brief Description Of The Figures Fig. 1: The flow chart of constructing plasmid pHEM3SGAC-LSO: Fig2: indirect immunofiuorescence detection of recombinant beer yeast INVSO/cHBM3SSAC-LSO(S-SP3Y Fig 5. Electrophoresis patiem of PCR identification of genstic stability of recombinant Lbeer yeast INVSo/pHiEMSGSAC-LSO(S-SPS3Y Fig. 4 immunofiuorescence assay of recombinant hear yeast INVSClpHBMIGBAC-LEO{ES-8P3) shows that the genetic generation expression bears fruit Fig 5: Pathological observation chart Fig. 6: Histoimmune chart; Fig. 7 immunonhistochermical detection of the distribution of PCVE infection in vivo, Description of the present invention The present invention will be further explained with reference to the following specific axamples, bul i is not kmited to this.

Embodiment 1 Construction of yeast engineering strain showing fusion proteins of LTB, SLT-leB and ORF2 to prevent porcine circovirus disease

4} According to yeast codon preference, the LTE gene, SLT-HeB gens and ORFZ AF 92 porcine circovirus coding nucleocapsid protein Cap in pathogenic Escherichia coll were modified; Designing primers needed io synthesize recombinant gene LSO, 2) The modified LTB, SLT-JleB and ORFZ are amplified by overlapping FOR technology, and the full length of LSO fusion gene is obtained by ligating the linker sequence; The nucleic acid sequence of LSG is shown in SEGIDNG.2; 3) Saccharomyces cerevisiae display expression vector pHEMSGSAC was digested by Cpol and Not I; 43 Introducing GTOA Into the § end of the fusion gene L3G, and introducing two nucieotide sequences of GGCCA into the 3 end The amplified product was treated by T40NA polymerase ai 12°C for 20min under the protection of dTTP, and LSO gens with sticky ends of Cop I and Not ! was obtained, 5) The fusion gene LSG is inserted into the vector pHEMSGSAC by DNAlgase to form the recombinant plasmid pHEMSSSAC-LSO, and the flow chart is shown in Fig. 1; 6) Plasmid pHEM3GSAC-LSO was Iinearized by Hpal digestion, the resistance gene and replication point of Escherichia coll were removed, and then transfected into competent cells of Saccharomyces cerevisiae InvSet by Hthium acetate chemical transformation, costed with URA-deficient medium SC {1.24% YNE, 2% glucose, 002% Trp, 002%Leu,

0.01% His, 1.5% Agar), and cultured at 25°C-30°C for 2-4 days to obtain the recombinant transformant INVSCHSHBMSSSAC-LSO.

Embodiment 2 Acquisition of 5-8P73 recombinant strain

1. Analysis of growth characteristics of 8-8P3 recombinant strain The recombinant strain S-SP3 was streaked on the solid YEPD plate, and single colony was selected into the liquid YNB-HLAD medium, After culturing at 286 and 200 min for 18 hours, it was inoculated into the yeast medium which had been sterilized in advance according to the inoculation amount of 3%, and the culture medium temperature was 28°C, oH was 4.6, and the centrifuging speed was 170 min.

Experiments prove that the genetic engineering strain of the invention can ferment at high density, and the OD6C0 is above 40, the whole process does not need to add inducers in the whole fermentation process, and can express efficiently ai the end of logartfmi 9 strictly. At last, the biomass of Saccharomyces cerevisiae reached 8.124g/L

2. Genstic stability of S-SP3 recombinant strain The S-5P3 recombinant strain prepared by the invention is crossed and cultured on a solid YEPD plate, single colony is selected and cultured in à liquid YNB-HLAD culture medium at 280 and SOOr/min for 16 hours, then transferrad to the YNB-HLAG liquid culture medium at a volume ratio of 1.1000 for 48 h, and then transferred to the YNB-HLAG liquid culture medium at a volume ratio of 1:1000 for continuous passage for 30 generations. Ses Fig. 3 and Fig. 4 for results, Fig. 2 is the electrophoresis pattern of genetic stability PCR identification of recombinant beer yeast INVSc/DHBEMOSBAC-LSO(S-GPSY Lane 1 empty vector control, 2:5 generations, 3:10 generations, 4:15 generations, 5:20 generations, 6:30 generations, M 2000. The results showed that the deleted strain could amplify 13082650 of the LEO fusion gens, and the vaccine strain was stable after 30 generations of YEPD culture, and would not return to strength. Sequence analysis of LSO genes of each generation of recombinant Gacteria showed that the sequences were 100% homologous, Moreover, immune fluorescence showed that the protein expression of each generation was stable.

Fig. 4 shows the genetic expression and fruiting of recombinant brewer's yeast INVSCpHBM3GBAC-LEO{E-8P3 by immuncliucrescence assay, and the sub-Fig 1 shows: 5 generations, 2 10 generations, 3: 15 generations, À 20 generations, 5 30 generations, and 6; empty vector bacteria control. The fluorescent results showed that the protein expression of each generation was stable.

Embodiment 4 Preparation of S-SP3 recombinant strain vaccine The obtained S-SP3 recombinant strain was identified, and each generation was inoculated on YEPD medium. The genetic stability of the recombinant strain was identified Ly POR detection with primers. After 20 passages, il was found that the fragments with sizes of 151600 were still amplified, and their genetic performance was stable, Westarn-blotting and immunofluorescence detection showed that the recombinant protein LSO could be stably expressed in the recombinant strain and had good immunogenicity.

The recombinant strain 5-SP3 was cultured in YEPD solid medium, and single cotsny 958 was selected and cultured in LE liquid medium until the viable bacteria concentration reached 1x10'"CFU/mL. Adding gelatin proteciant according to the volume ratio of bacterial solution to gelatin protectant of 7:1 (the preparation method of gelatin protectant is Adding 40g sucrose and 2g gelatin to 100m deionized water, fully melting it, sterilizing it at 121°C for 30min, and packing it in a sterilized freeze-dried bottle at 2 OmL/ bottle. Freeze-drying at 50°C, freeze-dry for 36-40 h, then capping it, dissolving with 10% aluminum gel physiological sait, count the viable bacteria (CFU), and make sure that thers is no contamination of miscellaneous bacteria, and store at -20°% for later use, 50 as to be used as a vaccine strain for developing recombinant epidemic vaccine.

Embodiment 55-8P3 recombinant strain vaccine safety evaluation

1. Safety test of non-target animals (CD-1 female mice) and pathogenicity test Five-week-oid female inbred line CD mice were inoculated with different doses (5x 10UCFU ~ 5x 1075 CFU) of recombinant bacteria and vector bacteria in the late logarithmic growth stage, and the clinical situation of sach group of mice was observed after inoculation.

The experimental design and results are shown in Table 1.

The results in Table 1 show that the highest dose, that is, the inoculation dose, is 50,000 times of the normal immunization dose (3«10'") of pigs. The mice in the experimental group and the control group are normal in performance and normal in pathological examination. The results showed that recombinant bacteria and vecior bacteria had no pathogenicity to mice and were safe to mice.

; ; a ; , ; ; LU501952

2. The infection route of the recombinant strain 8-5P3 of the present invention affecls the safety Five-week-old female inbred strain CDI mice were inoculaled with different doses (5x 10UCFU ~ 5x 10'5°CFU) of recombinant bacteria S-SP3 in the late logarithmic growth stage by intraperitoneal injection, subculansous injection and intragastric administration, respectively. The clinical situation of sach group of mice was observed after inoculation. The test design and results are shown in Table 2. Table 2 Pathogenicity of different inoculation routes È Vs | = N ı Intrapertongal | TTT TTT ayection : Ls | $ ' it | Héragainie a ss adromstrafion ie] The results in Table 2 show that different inoculation routes are not pathogenic to mice. 3, Safety evaluation of the recombinant strain S-SP3 of the present invention on pigs The optimal dose of recombinant strain S-SP3 was 1 5x10/7CFU. The recombinant strain S-5P3 prepared by the invention was inoculated to G pregnant sows and 6 weaned piglets according to the number of live bacteria injected into 1.5x10%CFU per pig, and the results are shown in Table 3 and Table 4. Table 3 Changes of body temperature and farowing of pregnant sows afer vaccination

Posen Temperature change after vaccinated _

DATE SD ID 7D 3D «D SD < p| NE | ne Table 414-d8y-0id piglets’ body temperature changes and growth after vaccination Temperature change after vacomated Rubel | Deyagot | DSB 2:59 RD #4 BP : D os : | os W808 ELE TN | Wierd TN TN TN ow iw D From Table 3 and Table 4, it can be seen that pregnant sows and piglets did not show any adverse reactions, their mental state and diet were normal, and their body temperature did not change much before and after vaccination, and there was no significant difference in farrowing performance between the pregnant sows in the experimental group and the control group (Table 3) The age of piglets reaching 9Dkg weight in the experimental group is 148 days, and that in the control group is 154 days, with no significant difference {Table 4}. The results showed that the vaccine was safe for pregnant sows and weaned piglets, Embodiment 6 Immune efficacy test of recombinant bacterial vacoine S-SPS prepared by the invention in pigs

1. Immunization program of pigs Twenty-four piglets, aged 20-25 days and negative for porcine circovirus disease, wers selected and divided into three groups. The first group was diluied water control group, numbered 1-8, the second group was carrier bacteria 8 control group, numbered 9-16, and the third group was S-SP3 recombinant bacteria vaccine group, numbered 17-24 ne 002 diluted control group, mb of diluent was injected into the neck muscle of each pig, while in the vector S control group and the recombinant vaccine S-SP3 group, tmb of culture {live bacteria content 1.5<10""CFU) was injected into the neck muscle of each pig, and immunization was carned out twice with an interval of 2 weeks.

Biood samples were collected two weeks after the first immunization and two weeks after the second immunization, and the antibody level of LSO recombinant protein was detected by commercial ORFZ ELISA kit à, Detection of ELISA antibody level in immunized piglets Two weeks after the first immunization and two weeks after the second immunization, blood was collected from the anterior vena cava, serum was separaled, the titer of LSO specific antibody was detected by indirect ELISA, and the average value was calculated. The results are shown in Table 5.

Table 5 Detection of serum antibody of piglets immunized with recombinant vaccine S-SP3 prepared by the invention (ELISA method) [eee SEE Group ofExpernment | Carrier bacteria comol gow er bacteria control Ecoup as es The results in Table 5 show that the specific antibody tier of the recombinant bacterial vaccine S-SP3 group of the present invention is 1: 480 two weeks after the first immunization, and the antibody titer of LSO rises to 1:2410 two weeks after the second immunization. However, the carrier bacteria S control group and diluent control group were negative (< 1:5). The above results show that the S-SP3 recombinant vaccine prepared by the invention can induce the body to produce specific humoral immune response of anti-Salmonelia and LSO specific antibodies after immunizing piglets.

Froiective test of piglet immunized with recombinant vaccine S-SP3 prepared by he Oo invention Three swing edema-negalive Z-week-old piglets immunized with the recombinant bacterial vaccine S-8P3 of the present invention and the control group (six non-infected piglets) were infected with porcine circovirus FCV23Z{4<107TCID50) intramuscularly on the 22nd day after immunization. As a result, the body temperature of the challenged pigs in the immunized group rose slightly within one day after the challenge, and then returned to the normal level, showing no other adverse reactions. All the infected pigs in the control group had fever reactions. No other clinical symptoms were found in all pigs. See Fig 5 and Fig. 6 for the results, Posi-mmunized pigs (Fig, SA, 5C) Except for a small amount of bleeding spots inthe lungs, other tissues are normal, the alveolar wall structure of the lungs is complete, and there is no foreign body in the alveciar cavity, Non-immunized control pigs (Figure 58, DB) pulmonary interstitial widened, bleeding spots of different sizes distributed on the surface, pulmonary interstitial pneumonia, alveolar cavity hemorrhage, alveolar wall congestion, vmphatic infiltration around bronchl, slightly enlarged spleen, infarcted focus on the edge, and a few bleeding spoils on the surface, The comparison of histopathological changes between vaccinated pigs and non-vaccinated pigs further shows that the vaccinated pigs have good resistance to the attack of large dose of PCVZ.

in the vaccine group, the structure of tracheai epithelial cells, cilia and tracheal glands is complete {Figure 8-4, 5-5), bronchioles and alvaeol are normal, and the skin structure is complete. In the empty carrier control group, the ciliary defect of tracheal epithelial cells, shight edema of alveol and slight thickening of skin epidermis were found. in some pig lungs, squamous matapiasia of tracheal epithelium is obvious, and tracheal cila are missing or falling off (Figure 6-0). The infiltration of neutrophils and lymphocyles can be seen in tracheal mucosa, and about 40% of tracheal serous glandular epithelium is metaplasia. Alveolar congestion, edema, and widening of pulmonary alveolar septum (Figure 8-0).

Four days after infection, the main organs and issues were collected for immunohistochemistry, and the distribution of virus in the body was detected, The results are shown in Table 8.

Table 6 Detection results of PCV2 in serum at different time after challenge

- LU501952 (Note: the numerator is the number of detected POVZ pigs, and the denominator is the number of pigs to be detected) From Table 5, à can be sean that after 3 weeks of immunization, only ong pig in the immunized group developed viremia, and no viremia was detected after 6 days of immunization, indicating that the vaccine can effectively protect pigs from PCVE attack and obviously shorten the time of viremia, POWV2 positive signals were detected in lungs, spleens, iymph nodes, kidneys and other tissues of non-challenged pigs (Figure 7). Figure 7 shows the distribution of PCV2 in vivo after immunohistochemical detection, in which A and 5 are lymph nodes; © is spleen, D is the lung: E and f are kidneys, This shows that the virus is addicted to related tissues, and further shows the pathogenicity of the virus to the organism. Al the same time, the related organs and tissues showed specific histopathological manifestations. Hf shows that the genetic engineering vaccine based on recombinant S-SP3 vaccine strain can protect the immune pigs against the attack of lethal dose strain.

The results of the invention show that the recombinant beer S-5P3 vaccine containing no resistance marker and expressing LTE, SLT-HeB and ORF2 gens fragments can stimulate the organism to produce high-valent antibodies against LTB, SLT-HeB and ORF2 immune antigens of porcine circovirus disease, and has low toxicity to mice and pigs and good safely. in the immune protection test, à has a high protection rate against porcing circovirus.

The above-mertioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limied by the above-mentioned embodiments. Any other changes, modifications, substitutions, combinations and simplifications made without departing from the sourit and principle of the present invention should be equivalent replacement methods, which are included in the scope of protection of the present invention.

Sequence list 10501952 <110> GuangZhou Wisdom Bio-Technology Co.

Lid.

Yangize University <120> Yeast enginesring Dacteria for preventing porcine CITCOVITUS diseass <130>PT2001 <1607>2 <170>Patentinversions.5 < 21074 <211>436 <212>PRT <2{3>artificial sequence <400>1 MetAsnlysValilLysCysTyrVaiLeuPheThrAlaLeuleuserSer 151015 LeuTyrAlaHisGivAlaProGinThrile ThrGluteuCyscerGii 202530 TyrArçAsnThrGiniie TyrThrileAsnAspLysiieLeuSerTyr 354045 ThrGiuSerMetAlaGiyLysArgGluMetValllellieThrPhelys 505580 SerGiyGluThrPheGinValGluValProGhySerGinhisHieAsp 85/07580 SerGinLyslysAlalleGluArgMeiL ysAspThrleuArglieThr 859085 TyrLeuThrGiuThrlyslisAspiysLeutysVaiTrpAgnAsnLys 100106110 ThrProiieSerleAlaAlalleSerMetGluAsnProAlaProAia 1158120128 FroAlaAspCysAlalysGlylLyslleGiuPheSerlysTyrAsnGlu 130135140 AspAsnThrPheThrVallLyvsValSerGiyAraGluTyrTroThrAsn 145150155160

AroTrpAsnlLeuGinProLeul euGinSerAlaGinleuThrGlyMet 10501952 1865170175 ThrValThrileleSerAsnThrCysSerSerGlySerGlyPhedls 180185190 GinvValLysPheAsnProAlaProAlaProMetThrTyrProArgArg 1952002058 ArgPheArgArgArgArghHisArgProArgSerbisLeuGlhyGinlle 210215220 LeuArgArgAroProTroLeuValhisProArgitisArotyrAraTrp 225230235240 ArgArgLysAsnGlyHePheAsnThrArgLeuSsrArgThrPheGiy 245250255 TyrThvallyslysThrThrValargThrProSerTrpAiaValdso 260265270 MetMietAroPheAsniieAsnAspPheLeuProProGhyGiyGiySer 2762802885 AsnProLeuThrValProPheGluTyrlyrArgiieArgLysVailvs 280285300 ValGluPheTrpProCysSerFroileThrGinGiyAspArgGiy Val 205310315520 GlyserThrAlaVailieL suAspAspAsnPheVaiThrLysAlaThr 325330335 AlateuThrtTyrAspProtyrValAsnTyrSerSerArgHisThriie 340345350 ThrGinProPheserTyrHisserArgTyrPheThrProLysProVal 355360365 LeuAspSerThrieAspTyrPheGinProAsnAsnivsArgAsnGin 370375380 LeuTroleuArgleuGinThrSerAlaAsnValAsphisValGiyLeu 385350385400

GlyThrAlaPheGiuAsnSerlleTyrAspGinAspTyrAsnileArg 10501952 4054104158 YaiThriMel TyrValGinPhefrgGiuPhelsnb aul ysAspProPro 4203425430 LEUASNPIOLYyS 435 <210>2 <211>1308 <212>DNA <213> artificial sequence <400>2 aigascasagigaaastgtiaigiittotiactoctigtigtoiicHotatgoicaiso gotaciccacasacistiacigaacioigtiolgaatacaggaacacicagatitatact120 gtaaacgacaagatacigicliatacegagiclatgycigutasaagagaaatgottate 180 ataacititaagictggigsaaciitceaagiigaagiicecaggticicancataiigat24 tclcaasagasggotatigasagastgasagacacitigegiatiactiatitgacagaasi accasastigacaaatigicigictogastsacsagaciccaatatctatigeigotatta6i ictatggasasaticcageiccagcaccagetgatigigeotsagoggaaaatagagtioticis20 aagtatsatosagatascac(itiactotasagthagogategigaatactogacisatd&0 agatggastiigeaaccatigtigeaaicigeicagiigaciggistgacigtiactateb40 atttccaatacoigoictiagegatictyggtitegoicaagtiaagittastccagatecat Gi geoaccaaligactaltecaagaagaagatitagsagacgiagacacagaccaagateoicacG50 ttgogtcasaliiicacgagaagaccttactiogigcatcogagacatagatatagçatgg” 20 cotagaaagaaiggtatettigacactagotigiciaggactittgoctatacagiasa/80 gaagactactottagaaciccatotigagotatagalatgaigagaticaacatcaacgati4D titttgecaccaggiguiggtictastecactzacigticeatiigaatatiacagaatil agaasagttasagiigaaititogecatgeiciccastaacIcaggotgataggagiotsonD gotagtacigeogtiattiiggaigatastiicgtisctaaageisctgeciigacatat 1020 galccatatoitsactatagetcisgocacactatiaciosacotitticitatcaiicti 080 agatatticactocassaccoigicitagac(ciactiaiigattac{ticagocaaacaati 140 azaagasatcaatigiguitgagatigoaaactagigctaacgtigateatgiiguatig1 200 10501952 gotacigetittgaasacictattiatgstcaguatiataacataagagtiacaatgtact 260 gticaatttagagaatttaacctaasggaceeiceatigaateccaaat 308

Claims (8)

1. Claims

   1. Genstically engineered bacierium is characterized in thal genetically enginesred bacterium contains the gene encoding the LEO fusion protein, and the sequence of the LSO fusion protein is shown in SEQUID NO: 1

   2. Genetically engineered bacterium, according to claim 1, is characterized in that the gens encoding LEO fusion protein is obtained by modifying LTB gene, SLT-HeB gene in pathogenic Escherichia coli and ORFZ gene of encoding viral nucleocapsid protein Cap according to codon preference of genetic engineering bactena, and itis obtained by ligating with linker sequence.

   3. Genetically engineered bacterium, according to claim 1, is characterized in that the engineering bacteria is baer yeast,

   4. Genetically enginesred bacterium, according to claim 3, is characterized in that the nucisic acid sequence of encoding the LSO fusion protein is shown in SEQIDNG: 2.

   5, Genetically engineered bacterium, according to claim 3, is characterized in that the LEO gene was transferred into the vector pHEM3SGASG to obtain the expression plasmid pHBM3GSAC-LSO.

   8. Genetically engineered bacterium, according to claim 3, is characterized in that the expression plasmid pHBM3SSAC-LSO was transferred into Saccharomyces cerevisiae 10 obtain yeast genstic engineering bactena,

   7. Application of the genetically engingered bacteria of claim 1 in prevention and treatment of porcine ciroovirus disease.

   8. Application of the genetic engineering bacteria of claim 1 in preparing genstic engineering vaccine of porcine circovirus disease.