LU502210B1 - CTB-Cap fusion protein, its coding gene and application - Google Patents

Abstract

This invention belongs to the field of genetic engineering in biotechnology pharmaceutical industry, in particular to a CTB-Cap fusion protein, its coding gene and application. The amino acid sequence of CTB-Cap fusion protein is shown in SEQ ID NO:2. The base sequence of the coding gene of CTB-Cap fusion protein is shown in SEQ ID NO:1. The CTB-Cap fusion protein of the present invention utilizes the advantages of Lactococcus lactis, including safety, nontoxicity, easy cultivation, high efficiency of foreign protein expression, and it does not need to purify the expressed product, etc. This invention expresses the CTB-Cap fusion protein with Lactococcus lactis, which provides a certain theoretical basis for the development of porcine circovirus vaccine.

Description

Description 0502210 CTB-Cap fusion protein, its coding gene and application Technical Heid The invention belongs to the field of genetic engineering in biotechnology pharmaceutical industry, particularly reiates to CTB-Cap fusion protein, its coding gene and application.

Background AL present, the most effective way Io prevent and treat Porcine circovirus 2 (PCV) is vaccine. POV2 can be transmitted through mucosa, so mucosal protection plays an important role in blocking Hs transmission. Al present, the listed POV2 subunit vaccines are developed by Boehringer-ingeiheim Company, AKZO-NOBEL Company and PCY developed by Schering Plough Company abroad, Domestic subunit vaccines developed by Wuhan Zhongbo, Yangzhou Youbang, Qingdao Yibang and Pleco are also available for the market. Commercial POVE vaccines are mainly inactivated vaccines and subunit vaccines, However, the inactivated vaccing is difficult to prepare, ids virus titer is low, the production cycle is long with high production cost, which cannot completely block the spread of POVZ Most subunit vaccines use Escherichia coli expression vector or baculovirus expression vector, although Escherichia coli is easy to culture, itis easy to form inclusion bodies, which incresses the purification cost. However, baculovirus expression vector is difficult to express and ids production cost is high. The immune responses induced by these PCVZ vaccines are mainly celular and humoral immunity, which ignores mucosal immunity. Therefore, vaccines that can stimulate the body io produce mucosal immunity have great potential in preventing and controlling PCV2. As a recognized food safely microorganism in the world, lactic acid bacteria have high protein yield with short culture period, it has low production cost and low immunogenicity, and can carry antigen to intestinal mucosa, which is & good hve carrer to stimulate mucosal immunity. However, the mucosal vaccine generally has weak immune response, short antibody maintenance time and unsatisfactory mmune effect, so the use of effective mucosal immunopotentiator is particularly important, According to the concept of immunogenicity rimmune enhancement”, the PCV2 gens and Gholeratoxin B subunit (CTB) gene are linked in series, the recombinant Lactococcus lactis capable of expressing CTB-Cap fusion protein was constructed by genetic engineering technology, and the mmunogenicity of recombinant Lactococcus lactis a 0 verified by animal experiments, so as to lay a theoretical foundation for the research of recombinant live vector vaccine of POYZ.

Summary This invention uses the advantages of CTB-Cap and Lactococcus lactis, and provides CTB-Cap fusion protein, iis coding gene and application.

This invention provides the following technical solutions: CTE-Cap fusion protein, ds amino acid sequence is shown in SEQ 1D NO-Z, This invention further provides the application of CTB-Cap fusion protein in preparing COTCHMS circovirus type 2 Vaccine, This invention further provides the preparation method of CTB-Cap fusion protein is characterized by comprising the following steps: construction of expression vector the recombinant plasmid pNZ8148-CTB-Cap containing CTB-Cap and lactic acid bacteria expression vector PNZS148 was synthesized in the whole genome, and the clones containing the gene sequence of CTE-CAF were screened out by double enzyme digestion and PCR detection, construction of Lactococcus lactis expression system: the ohiained recombinant plasmid pNZ8148-CTB-Cap was transferred into lactic acid bacteria compatent NZ9000 by electrotransformation; the transformed bacterial suspension was spread on a GM17 piste containing chloramphenicol, and cultured at 30°C; single clones were selected, and the CTB-Cap fusion protein was induced to express CTH-Cap fusion protein with different concentrations of Nisin, and the baclerial cells were disrupted by sonication; positive clones were confirmed by SDS-PAGE and Western blot, The encoding gene of CTE-Cap fusion protein, its base sequence is shown in SEQ ID

NOT in the present invention, the coding gene of the CTB-Cap fusion protein is composed of the cDNA sequence coding CTB, the cDNA sequence of porcine circovirus type 2 Cap protein, six histidine cDNA sequences and the cDNA sequences of restriction endonucleases Neo | and Hind AM} sites, as well as the gens sequence conforming to the expression preference of Laciococous lactis, The CTB-Cap fusion protein of the present invention utilizes the advantages of Lactococcus lactis, including safety, nontoxicity, easy cultivation, high efficiency of foreign protein expression, and it does not need io purify the expressed product, etc. This ner 0 expresses the CTH-Cap fusion protein with Lactococcus lactis, which provides a certain theoretical basis for the development of porcine circovirus vaccine.

Brief Description Of The Figures in order to more clearly explain the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that nesd to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to these drawings without any creative effort.

Fig. 1 shows the identification results of recombinant plasmid pNZ8148-CTB-Cap. In the two figures, À results of positive plasmid restriction enzyme digestion, B: results of positive colony POR identification. Fig. 1: Marker, 2: CTB-Cap plasmid, 3. plasmid after double enzyme digestion, À positive CTB-cap colony.

Fig. 2 shows the identification results of CTE-Cap fusion protein, A SDS-PAGE analysis, B.Western biol analysis (anti-CTB) C: Western blot analysis (anti-his), Fig AM: protein molecular quality standard, 1: uninduced recombinant lactic acid bacteria, 2:30ngimi induced for 4h, 3:50 ng/ml induced for 4h, 4.100 ng/ml for 4h, 5:20 ng/ml for 4h, &:50ngfmi for 4h, 7100 no/mi induced 4h crushing and precipitation.

Fig, 3 shows standard curve of protein concentration determination.

Fig. 4 shows changes of body weight in each treatment group.

Fig. 5 shows specific antibody level in serum of mice immunized with CTB-cap fusion protein.

Description of the present invention in order to make the object, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, but not ail of them, Based on the embodiment of the present invention, all other embodiments obtained by ordinary technicians in the field without creative labor belong to the scope of the present invention.

1, the reagent and its preparation

{1 LE liquid culture medium: 19 pepione, 0.59 veast powder and 19 NaCl ara 0 dissolved in 100ml dH0 and sterilized under high pressure, (2) GM47 liquid culture medium: 4720 GM17 is dissolved in 100mL dH and sterilized under high pressure, {2} GM17 solid medium: 4.729 GM17 and 1.5¢ agar powder were dissolved in 100mL dH:0 and sterilized under high pressure: (3) GM17 solid medium: 4.729 GM17 and 1.59 agar powder were dissolved in 100mL dH>0, and sterilized under high pressure, (4) GMMS recovery medium: 4.72g/100mL GM17, ZOmM MgCh and 2mM CaCl are mixed evenly; (5) Electrophoresis buffer. 3.03g Tris, 18.77¢ glycine and 1g SDS were dissolved in 1000m1 dH; (6) Membrane transfer buffer. 5.8¢ Tris, 2.99 glycine, 0.379 SDS and 200ml methanol are dissolved in 1000mL dH; (7) TBST: 44g NaCl, mb Tris-HCY{PH=7,5) and 25001 Tween-20 are dissolved ir SOOmL dH20O

2. Acquisition and identification of recombinant plasmid pNZB8148-CTB-Cap Searching the gene sequences of CTH (JF274975.1) and POV2 Cap (DO235696. 1) and the expression vector pNZ8148 that NCSI has landed an. After optimization, they are sert to Zoonbio for full genome synthesis, and the obtained recombinant plasmid pN/8148-CTH8-Cap was transformed to MC1061 clone strain, picking positive clones to expand culture, and extracting recombinant plasmids, The recombinant plasmid was identified by double digestion with Noo | and Hind IH at 37°C for 3h the recombinant plasmid was detected by PCR using primers (F: d'ACGOGAGCATAATARAACGG 3, R: E'UGARMAGCGAAATCAAACGA 3) It can be seen from Fig 1-AÀ that the recombinant plasmid pNZ8148-C75-Cap was successfully constructed.

3. Construction of expression vector Mixing 1 ul of the correctly identified recombinant plasmid pNZ81485-CTB-Cap and 40 uL of lactic acid bacteria competent NZ9000 gently and evenly, then adding tio a 0.2 om electroporation cup in an ice bath, setting the electric shock parameters to 2000, 25uF, 2000, placing the electric rotor cup in the electric shock tank and shock immediately after the electric shock, adding 1 mL of GMMC recovery medium, ice bathing for 5 Ar 2210 transferring all the liquid in the electric rotor cup into a 1.5 mb EP tube, and incubating at °C for 1 hy taking 10 ub, 100 pb and 800 uit respectively on GM17 solid culture plaies containing 10 ug/ml chioramohenicol After the bacterial guid is completely absorbed, culturing at 30°C until transformants appear. The transformants were picked for PCR detection Using primers {F: S'ACGOGAGCATAATAAMAACSS 3 R: B'EGAAAGCOAAATCAAACGA 3). # can be seen from Fig 1-B that pNZ8148-CTB-Cap was transferred into Lactococcus lactis, and the recombinant Lactococcus acts was successfully constructed,

4. Expression of CTB-Cap fusion protein Picking the correctly identified transformants and inoculating in 5 mb of GM17 liquid medium containing 10 uc/mb chioramphanicol, and cultivating overnight at 30°C at 285 rom; inoculating the above-mentioned culture medium in 100 mL of GM17 liquid medium containing 10 pg/ml chloramphenicol, cultivatina at 30°C at 225r0m io a bacterial OD600 of about 0.4; taking 1 mb of culture medium, centrifuging at 10,000 rpm for 2 min at room temperature, discarding the supernatant, and resuspending the bacterial pellet with 1x loading buffer, adding Nisin to the remaining culture medium to the final concentration of 30, 50, and 100 ng/ml. and incubating at 30°C for 4h, inducing the expression of CTB-Cap fusion protein, and screening the best induction concentration, after the induction, 1 mL of culture solution was taken out, centrifuged at 10,000 rom for 2 min at room temperature, the precipitate was washed twice with PES, and then resuspended, the resuspended bacterial solution was ultrasonicaily disrupted, after sonication, the supernatant and the sediment were added to the loading buffer and resuspended. All samples obtained above were subjected to SDS-PAGE and Western Biot detection. it can be seen from Fig 2 that the recombinant Lactococcus lactis successfully expressed the CTB-Cap fusion protein after being induced by Nisin, and 30ng/mb, SüngimL and 100ng/mbl Nisin could induce the recombinant Lactococcus lactis to express the fusion protein. On the basis of the uniform loading amount, the amount of protein induced by Nisin concentration of 50ng/ml and (COng/ML has no difference, and the protein induced by 30ng/mb nisin is mors than that of 30ng/mib nisin Therefore, S0ng/mL Nisin is selected as the best induction concentration.

5, Determination of CTB-Cap fusion protein yield

Diluding the recombinant Cap protein at a concentration of 0.023 ma/mi as Faliowa 902210 inducing 100 mL of recombinant Lactococcus lactis to express the CTB-Cap fusion protein. After the expression, the cells were collected by centrifugation ai 10,000 rpm at room temperature for 2 min, and the precipitate was washed twice with PBS. The pellet was resuspended with tmL of PBS and then sonicated to oblain a suspension containing CTB-Cap fusion protein, which was diluted 1.10000 with PBS.

The mouse-derived anti-Cap protein monoclonal antibody was diluted 1500 with the protein coating solution, and then costed on the ELISA plate, 100 ul per well, standing overnight at 4°C: the costing solution was discarded, and each weil was washed iwice with 300 ul PEST, bmin per time; adding 200ul of 5% nonfat milk powder to each well, sealing at 37°C for 15h, discarding the blocking solution, adding 100 pl of the dilution containing CTB-Cap fusion protein and the recombinant Cap protein diluted according to the above table to each well, one replicate per well, and incubating at 37 °C for 1 hy discarding the protein dilution, and washing each well with 300 ul of PBST 4 times, Smin each time, adding 100 ul of HRP-labeled circular antibody that is diluted 1.10000 in blocking solution to sach well, incubating at 37°C for 30 min; discarding the circular antibody, washing each well with 300 yl PBST 4 times for 5 min each time, adding 100 ul TMB color developing solution io each well | and incubating at 37°C for 10 min in the dark; 50 pl of ELISA stop solution was added to each well to terminate the reaction, and the OD450 absorbance value was detected by a microplate reader. The OD value of the suspension containing the CTE-Cap fusion protein at 450mm was 1.545925 from the standard curve of the protein concentration determination shown in Fig 3 and the microplate reader. ft can be calculated that the expression of the CTB-Cap fusion protein is about 50 5mag/100mL.

8. Mouse immune protection test

6.1 Test grouping and processing Positive vaccine test group. porcine circovirus type 2 genetic subunit vaccins {derived from Escherichia col) 50 ulipisce; 25x10? test group: 25uL (10 10 CFL recombinant Lactococcus lactis; 5x10° test group: 25 ul (10 9 CFU) of recombinant Lactococous lactis; 510° test group: 25 pl {10 8 CFU) of recombinant Lactococcus lactis, PBS test group: 25 ul PBS.

25 female BALB/C mice of 6-week-0id were randomly divided into 5 groups with 5 fea 210 in each group. According to the above grouping, the mice in the positive vaccine test group were injected with 50 ub of the positive vaccins through the leg muscles, the 2.5210" test group, 5x10 test group and 5x10% test group were immunized with 25 ul of 2.5x101°CFY, 5x10°CFÜ and Sx10%CFU recombinant Lactococcus lactis by subcutaneous injection on the back, respectively. The PBS test group was subcutaneously infected with 25 ul of PBS through the back as a control, Except the positive vaccine group was immunized once, the other groups were immunized twice with an interval of 2 weeks.

6.2 Effects of recombinant Lactococcus lactis on weight gain in mice During the experiment, the body weight of the mice was recorded weekly. As can be seen from Fig 4, there was no significant difference in body weight between the groups {p=0.05}

6.3 Detection of anti-POV2E Cap-specific igh in mouse serum 14, 21, 28, 35, and 42 days after the first vaccination, Dicod was collected from the retro-orbitai venous plexus of mice, and the serum was separated, Ciluied with 1% BSA-PEST blocking solution, and the level of POV Z-specific antibody was determined.

After the recombinant Gap protein was diluted to 0.1 mom with the coating solution, 100 ul per well was used to coat the ELISA plate, standing overnight at 4°C; the costing solution was discarded, and gach well was washed three times with 300 pb PEST for 5 min gach time, 200 ul per well was added 1% BSA-PEST, sealed ai 37°C for 3 hours; discarding the blocking solution and wash each well with 300 ul PBET for 3 times 5 min sach time, adding 100 ui of diluted serum to gach well, one replicate per well and incubating at 37°C for 1 h; discarding the serum and wash each well with 300 pl FEET for à times, 5 min each time, adding 100 ul of blocking solution 1.10000 diluted gost arti-mouse HRP-ioG secondary antibody to each well, incubating at 37°C for 30 min, discarding the secondary antibody, washing each well with 300 yl PBST 3 times, 5 min gach time; adding 100 ul of TMB chromogenic solution to each well, incubating at 37°C for min in the dark; adding 50 ul of ELISA stop solution to sach well to stop the reaction, the 0D450 absorbance value was detected by a microplate reader. As shown in Fig, 5, 21d and 28d after the first immunization, the 2.510 experimental group showed an increasing rend compared with sach experimental group, but compared with 5x 10° test group, 5=105 test group and PES group, there was a significant difference (p>0.05). 35d and 42d a 0 the first vaccination, compared with the PBS group, the 25x10" experimental group was significantly increased {p<0.08), and the 5x10° experimental group and the 5x10° experimental group were significantiy diferent (p>0.05) The constructed recombinant Lactococcus lactis was immunogenic by SUDCUISNGOuS immunization.

7. Data analysis The data were determined by One-way ANOVA in GraphPad Prism ™ 5 {GraphPad Software, Inc California, USA) data analysis software to determine whether there was any difference between the data of sach group, and the results were expressed as Meantstandarderrors mean (SEM).

The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited to this. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, should be included within the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Sequence list 0502210 <*10> Shand Agricultural University <t20> CTR-Cap fusion protein, its coding gene and application <130> PT2136 <180>2 <2i0>1 <211>» 1289 <212>DNA <213> Artificial Sequence <220> <222> CTB-Cap fusion protein <40G>1 AGGAACTACA AAATAAATTA TAAGGAGGUA CTCACCATGG GCAAMAAAAAA GATTATOTCA 80

GOTATTTTAA TGTCTACAGT GATACTTTCT GOTGCAGCCO COTIGTCAGG TOTTTACOOT 120

ACCTATCCAA GAAGAAGATA TOGTAGAGOT CGTCATCGTC CAAGATCACA TCTTGGTCAA 180

ATTTTACGTS GTCGTCOTTG GTTATTACAT CCAAGACATA GATATCGTTG GCGTAGAAAA 240 AATGGTATIT TTAATACCCG TOTTAGCOGT ACATITTGGAT ATACTATTAA GCGTACCACC 300 GTTAAAACCC CTTCTTGOGE TOTIGATATS ATOAGATTCA ATATTAATGA TITTCTTCCA 360

COAGOTOGTG GATCTAATCC AAGATCTOTT CCATTTGAAT ATTATCGTAT TCGTAAAGTC 420

AAAGTCGAAT TTTGOCCATG TTCACCTATT ACACGAAGGOTG ACCOTGGAGT TGGTTCATCA 480 GOTGTTATTT TAGATGATAA TTTTGTCALC AAAGOTACCG CATTAACTTA TGATOCATAT 540 GTTAATTATA GUAGCCGTCA TACTATTACC CAACOTTITI CATATCACAG CAGATATTTT 800 ACCCCAMAAC CAGTTUTTOA TAGCACTATT GATTATITIC AACCAAATAA TAAGCGTAAT 560

DAACTITGSC TTOSTOTTCA AACTGOTGOA AATGTTGATE ATGTIOGATT AGGTACTGCA 720 TTTGAMAATA GCATATATGA TCAAGAATAT AATATTCGTG TCACCATGTA TOTCCAATTT 780

CGTGAATTCA ATCTTAAAGA TCCACCACTT AATCCAGGTG GAGGTGGTTC TGGTGGABOI 0 840

GGATCAGGASG GTGGTGGTTG AACTGOTCAA AATATTACASG ATOTTTGTGG CGAATATOAC 500 AATACACAAA TATATACCOT TAATGATAAA ATTTTTAGCT ATACCGAAAS COTTGCCGGT 960

AAAAGAGAAA TGGOCAATTAT TACTTTTAAA AATGOTGCTA TTTTTCAAGT CGAAGTCCCA 1020

GGTTGTCAAC ATATTGATTC ACAAAAGAAA GCOTATTGAAC GTATGAAAGA TACCOTICOT 1080

ATTGOTTATC TTACCGAAGC TAAAGTTOAA AAACGTTTGTG TOTGOAATAA TAAGACCCCA 1140

CATGOCAATTG CTOCTATITC AATGOCAAAT CATCACCATC ATCATCATTG ATCTAGAGAG 1200

CTCAAGCTTT CTTTGAACTCA AAATTAGAAA AUCAAGGOTT GAAACOTTCA ATTGAAATGG 1260 CAATTAAACA AATTACAGCA COGTGTTGOGT 1289 <210>2 <211>284 <242>PRT <213>Artificial sequence <220> <223>CTB-Cap fusion protein «4002 MOKKEHSA LMSTVILSAA APLSGVYATY PRRRYRRRRH RPROHLGOI, RRRPWLLHPR 60 HRYRWRRKNG IFNTRLSRTF GYTIKRTTVI TPSWAVDMMR FNINDFLPPG GOSNPROVEF 120 EYYRIBKVKY EFWRCSPITG GDRGYGSSAY ILDDNFYTKA TALTYDPYVN YSSRHTITOP 180 FSYHSREYFTP KPVLDSTIDY FOPNNKRNQL WLRLQTAGNY DHVGLGTAFE NSIYDQEYNI 240 RYTMYVOFRE FNLEKDPPLNP GGGGSGSGGS GGGOSTFONI TOLCAEYHNT QIYTLNDKIF 300 SYTESLAGKR EMAHTFKNG AIFOVEVPGE GHIDSGKKAT ERMKDTLRIA YETEAKVEKL 380 CVWHRNKTPHA TAAISMANHM HHMM 384

Claims (4)

Claims LU502210

1. CTE-Cap fusion protein, ils amino acid sequence is shown in SEQ ID NG2.

2. According to claim 1, the application of CT E-Cap fusion protein in preparing porcine CHCOVirus type 2 vaccine,

3. According to claim 1, the preparation method of CTB-Cap fusion protein is characterized by comprising the following steps. construction of expression vecior. the recombinant plasrid pNZ8148-CTB-Cap containing CTB-Cap and lactic acid bacteria expression vector PNZ8148 was synthesized in the whole genome, and the clones containing the gene sequence of CTB-CAP were screened out by double enzyme digestion and PCR detection, construction of Laciococeus lactis expression system: the obtained recombinant plasmid pN281486-CTB-Gap was transferred into isctic acid bacteria competent NZ9000 by slectrotransformation; the transformed bacterial suspension was spread on a GM17 piste containing chioramphenicol, and cultured at 30°C, single clones were selecied, and the CTB-Cap fusion protein was induced to express CTB-Cap fusion protein with different concentrations of Nisin, and the bacterial cells were disrupted by sonication; positive clones were confirmed by SDS-PAGE and Western blot.

4. The encoding gene of CTB-Cap fusion protein, its base sequence is shown in SEQ 1D NOT