CN104120142B - A kind of secreting, expressing PEDV cAg COE Protein reconstitution Lactococcus lactis and its preparation method and application - Google Patents
A kind of secreting, expressing PEDV cAg COE Protein reconstitution Lactococcus lactis and its preparation method and application Download PDFInfo
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Abstract
The invention belongs to animal organism pharmaceutical engineering field, specifically disclose a kind of secreting, expressing PEDV cAg COE Protein reconstitution Lactococcus lactis and its preparation method and application. The present invention is by design many primers, the method of Overlap extension PCR is adopted the sequences such as signal peptide to be connected on the gene of PEDV cAg COE, and it being connected to Lactococcus lactis expression vector pNZ8048, electricity is transformed in Lactococcus lactis NZ9000 cell to obtain restructuring bacterium. Bacterium of recombinating obtains with nisin induction expresses, full culture after induction directly can stimulate mouse as oral vaccine and cause stronger cellullar immunologic response, this Recombinant Lactococcus lactis can as a kind of new oral vaccine product with good industrial prospect, the harm of pig industry being played a positive role to alleviating PEDV, promotion pig industry being developed in a healthy way has important practice significance.
Description
Technical field
The present invention relates to animal organism pharmaceutical engineering field, more specifically, it relates to a kind of secreting, expressing Porcine epidemic diarrhea virus cAg COE Protein reconstitution Lactococcus lactis and its preparation method and application.
Background technology
Porcine epidemic diarrhea virus (PorcineEpidemicDiarrhea, PED) it is one of worldwide pig transmissible disease, within 1978, being reported first in Britain, multiple areas eruption and prevalence in the world comprising China, causes heavy losses to various countries' pig industry subsequently. The outburst of PED in Asia causes extremely high mortality ratio, and when having research display Japan 1993��1994 years outburst PED, 5 pig farm piglet death toll are just more than 10,000, and mortality ratio is 30%��60%; It is reported from February, 2010 in November, 2011, the piglet death toll that China outburst PED causes is more than 50,000, and mortality ratio is up to 90%��100%. 2013, the U.S.'s also reported first existence of PEDV, 2014 years continued outburst with popular.
PEDV until today, is not only effectively controlled after being found, has become to affect the great virus of whole world pig industry on the contrary, has caused huge financial loss. At present for the prevention mainly vaccination of PED, although the deactivation vaccine of domestic PEDV and attenuated live vaccines obtain promotion and application widely, decrease the sickness rate of PED to a certain extent, but still cannot control completely. From at present both at home and abroad to result of study and the effect of PEDV; piglet obtains female source sIgA mainly through colostrum; producing the passive immune protection to coronavirus property diarrhoea, the circulating antibody IgG in serum can not provide protection, is also exactly can not produce the immunity of female source through non-bowel immunity. For the quick variation of PEDV and intestinal mucosa immunity feature, constantly development safety, can seem particularly important by the oral new generation vaccine immune effect that improves vaccine.
The part S glycoprotein that the part protective antigen gene (COE gene) of PEDV encodes has antigenicity, it is possible to induction body produces neutralizing antibody. Owing to the immune effect of serum antibody is undesirable, the circulating antibody (sIgA) that intestinal mucosa immunity produces is the potent antibodies that opposing PEDV infects. Therefore, select safety non-toxic, it is possible to the carrier system of antigenic substance also can be expressed and transmit to survival in enteron aisle, and stimulating mucosal immunity system generation mucosal immune response is significant to the control of this disease effectively.
Lactococcus lactis (Lactococcuslactis) is the most important and the most typical kind in lactococcus, is widely used in the food industry, is acknowledged as safe (generally-regarded-as-safe, GRAS) food-grade microorganisms. Using Lactococcus lactis as host's bacterium expressing heterologous albumen and enzyme, become the focus of foodstuffs industry, bio-pharmaceuticals and vaccine research gradually.
Summary of the invention
Technical problem to be solved by this invention overcomes defect not good for PEDV immune effect of vaccine in prior art, it is provided that the preparation method of a kind of secreting, expressing PEDV cAg COE Protein reconstitution Lactococcus lactis.
2nd object of the present invention is to provide the secreting, expressing PEDV cAg COE Protein reconstitution Lactococcus lactis that aforesaid method prepares.
3rd object of the present invention is to provide described recombinant lactic acid bacteria and is preventing the application in porcine epizootic diarrhea as oral vaccine.
4th object of the present invention is to provide a kind of prevention porcine epizootic diarrhea oral vaccine.
5th object of the present invention is to provide the preparation method of above-mentioned prevention porcine epizootic diarrhea oral vaccine.
In order to realize above-mentioned purpose, the technical scheme of the present invention is design many primers, the method of Overlap extension PCR is adopted the sequences such as signal peptide to be connected on codon optimized COE gene, realize the secreting, expressing of COE albumen with Usp45 signal peptide, realize the increase of expression amount with enhancement sequences (LEISSTCDA) and on position thereof.
The present invention is achieved by the following technical programs:
The preparation method of a kind of secreting, expressing Porcine epidemic diarrhea virus cAg COE Protein reconstitution Lactococcus lactis is provided, it is characterised in that, comprise the following steps:
S1. the COE gene of synthetic after codon optimized, designs many primers, adopts the method for Overlap extension PCR to be connected on COE gene by Usp45 signal peptide sequence;
S2. the COE gene that S1 obtains is connected to pNZ8048 and screens and obtain recombinant plasmid pNZ8048-COE; Adopt electrotransformation to import in Lactococcus lactis L.lactisNZ9000 by pNZ8048-COE recombinant plasmid, obtain Recombinant Lactococcus lactis L.lactisNZ9000/pNZ8048-COE;
COE gene order after codon optimized described in S1 is as shown in SEQIDNO:1, and each primer sequence is as shown in SEQIDNO:2��9; The primer sequence of described screening recombinant plasmid is as shown in SEQIDNO:10��11.
Overlap extension pcr (genesplicingbyoverlapextensionPCR, it is called for short SOEPCR) owing to adopting the primer with complementary end, PCR primer is made to define overlapping chain, thus by the extension of overlapping chain in amplified reaction subsequently, the amplified fragments overlap of different sources is stitched together. This technology utilizes round pcr can carry out efficient gene restructuring in vitro, and does not need endonuclease digestion and ligase enzyme process, this technology can be utilized to obtain other very soon and rely on the method for digestion with restriction enzyme to be difficult to the product obtained. In the prior art of PEDV vaccine, also do not find to utilize overlap extension pcr, and obtained the expression system of Lactococcus lactis by the goal gene through optimizing and enhancement sequences etc. Overlap extension pcr is successfully it is crucial that the design of overlapping complementary primer, and contriver, in the know-why of Overlap extension PCR, is groped by a large amount of experiment research and condition, obtains above-mentioned many primers that can be used for efficient amplification.
Preferably, the response procedures of Overlap extension PCR described in S1 is: 98 DEG C of denaturation 5min; 98 DEG C of sex change 10s, 55 DEG C of annealing 10s, 72 DEG C extend 5s, 30 circulations; 10min is extended after 72 DEG C.
Preferably, electrotransformation described in S2 is: get L.lactisNZ9000 competent cell, adds pNZ8048-COE recombinant plasmid, mixes even and proceeds to electricity and transform cup, adds recovery media, quiescent culture after ice bath after electric shock, plate screening height copy transformant.
Lactic acid bacteria expression system has the following advantages: tight type expression system, strictly controlled, does not almost express when non-induced; The extracellular protease of Lactococcus lactis is little, and the exocytosis mediated by signal peptide expresses the degraded that can eliminate intracellular protein enzyme to a certain extent, increases stability and the output of target peptide.
As a kind of optimal way, the preparation method of above-mentioned secreting, expressing Porcine epidemic diarrhea virus cAg COE Protein reconstitution Lactococcus lactis comprises the steps:
S1. the structure of secretor type recombinant plasmid pNZ8048-COE
S11. the codon preference optimization of related gene sequence and synthesis: by COE gene, Usp45 sequence and the equal codon optimization of other modified dna sequence, the on position of selective enhancement sequence simultaneously, design many primer NcoI-Usp1, Usp2, Usp3, Usp4, Usp5, Usp6-COE-F, COE-F, COE-His-HindIII-R, have also been devised primer pNZ1 and pNZ2 of the PCR detection for recombinant plasmid and order-checking simultaneously, the NcoI-Usp1 of optimum synthesis, Usp2, Usp3, Usp4, Usp5, Usp6-COE-F, COE-F, COE-His-HindIII-R, pNZ1, pNZ2 primer sequence is respectively as shown in SEQIDNO:2��11,
S12. the pcr amplification of COE gene fragment modified without upstream: taking containing the plasmid of the COE gene of optimum synthesis as template carries out pcr amplification;
The Overlap extension PCR amplification procedure of the complete modification fragment of S13.COE gene: the PCR primer reclaimed in S12 is as template, add high-fidelity DNA polymerase 2 �� PrimeSTARMix25 �� L, the each 1 �� L of primer NcoI-Usp1, Usp2, Usp3, Usp4, Usp5, Usp6-COE-F, COE-His-HindIII-R, template 0.5 �� L, uses ddH2O mends to 50 �� L, and PCR response procedures is: 98 DEG C of denaturation 5min; 98 DEG C of sex change 10s, 55 DEG C of annealing 10s, 72 DEG C extend 5s, 30 circulations; 10min is extended after 72 DEG C;
S14. the structure of recombinant plasmid pNZ8048-COE: the PCR primer NcoI reclaimed in S13, HindIII are carried out double digestion process, in the same way pNZ8048 empty plasmid is carried out double digestion, product Transformed E .coliMC1061 competent cell will be connected by ligase enzyme, detection obtains positive plasmid, is and obtains recombinant plasmid pNZ8048-COE;
S2. the structure of the secretor type Recombinant Lactococcus lactis of COE gene is contained
S21. the preparation of Lactococcus lactis Electroporation-competent cells: frozen L.lactisNZ9000 Lactococcus lactis is recovered, get 30 �� LL.lactisNZ9000 competent cells, ice bath melts, adds 1 �� LpNZ8048-COE recombinant plasmid, mixed even gently; Above mixture is proceeded to the electric shock cup of ice precooling, give rapidly a monopulse, optimum configurations is 2kV, 25 �� F, 200 ��, softly add the recovery media G-M17MC of 1mL ice precooling immediately, bacterium liquid are all sucked a sterile centrifugation tube after electric shock, cover tightly pipe lid, 30 DEG C of quiescent culture 1��1.5h after ice bath 5min; Bacterium liquid is coated with and chooses and get single bacterium colony Screening and Identification and obtain positive recombinant expressed bacterium called after L.lactisNZ9000/pNZ8048-COE;
Preferably, after the on position of enhancement sequences described in S11 is Usp45 signal peptide, before goal gene COE, being in centre, position is closed and is: (PnisA::SPUsp45:: LEISSTCDA::COE), wherein PnisA is promotor, the aminoacid sequence that " LEISSTCDA " is enhancement sequences.
The recombinant plasmid pNZ8048-COE that offer aforesaid method prepares and recombinant lactic acid bacteria galactococcus L.lactisNZ9000/pNZ8048-COE.
The application of above-mentioned Recombinant Lactococcus lactis L.lactisNZ9000/pNZ8048-COE in preparation prevention porcine epizootic diarrhea oral vaccine is provided.
A kind of preparation method preventing porcine epizootic diarrhea oral vaccine is provided, comprise the steps: Recombinant Lactococcus lactis L.lactisNZ9000/pNZ8048-COE is inoculated in G-M17C liquid nutrient medium, quiescent culture spends the night, get overnight culture and it is inoculated in G-M17 liquid nutrient medium with certain proportion, continue to be cultured to bacterium and enter logarithmic phase, adding inductor Nisin to final concentration is 1��2ng/mL, and induction certain time, the full culture after induction is directly as oral vaccine.
Preferably, the temperature of described quiescent culture is 30 DEG C.
Preferably, described certain proportion is inoculated as recombinant expressed for L.lactisNZ9000/pNZ8048-COE bacterium is inoculated in G-M17C liquid nutrient medium with the ratio of 1:100.
Preferably, described logarithmic phase is the OD value of described inoculum is 0.4��0.6.
Preferably, described induction certain time is 3��6h.
As a kind of optimal way, the preparation method of above-mentioned prevention porcine epizootic diarrhea oral vaccine, comprising the steps: with the ratio of 1:100, recombinant expressed for L.lactisNZ9000/pNZ8048-COE bacterium is inoculated in G-M17C liquid nutrient medium, 30 DEG C of quiescent culture spend the night; Overnight culture is inoculated in 10mLG-M17C liquid nutrient medium with the ratio of 1:50, continues to cultivate about 2.5h and enter logarithmic phase (OD to bacterium600=0.4��0.6); Being divided into induction group and do not induce group, it is 1 or 2ng/mL that induction group adds inductor (Nisin) to the final concentration of 1 �� g/mL, terminates cultivating after induction 3��6h, and the concentration adopting gradient dilution coated plate method to measure restructuring bacterium reaches 1012The CFU/mL order of magnitude, the full culture after induction can directly as oral vaccine.
The oral vaccine of the prevention porcine epizootic diarrhea that offer aforesaid method prepares.
Preferably, above-mentioned oral vaccine can adopt gavage or the mode fed to use.
Compared with prior art, the present invention has following useful effect:
The present invention provides the preparation method of a kind of secreting, expressing Porcine epidemic diarrhea virus cAg COE Protein reconstitution Lactococcus lactis. Namely L.lactisNZ9000/pNZ8048 lactic acid bacteria expression system is adopted, and add the secreting, expressing that Usp45 signal peptide and enhancement sequences etc. carry out PEDV cAg gene C OE gene, inductor Nisin is the class antimicrobial peptide produced by milk-acid bacteria, there is efficient, safe, nontoxic characteristic, add the probiotic properties of milk-acid bacteria as probiotic bacterium, this lactic acid bacteria expression system is made to become a food grade expression system, it is possible to take together with thalline.
It is used for the Lactococcus lactis prepared expressing the vaccine of cAg COE albumen preparation for PED, compared with vaccine existing with market, produce to have superiority with maintaining in intestinal mucosa IgA antibody, full culture after induction directly can stimulate mouse as oral vaccine and cause stronger cellullar immunologic response, this Recombinant Lactococcus lactis can as a kind of new oral vaccine product with good industrial prospect, the harm of pig industry being played a positive role to alleviating PEDV, promotion pig industry being developed in a healthy way has important practice significance.
Accompanying drawing explanation
Fig. 1 is the pcr amplification result of the COE gene fragment modified without upstream; M is DL2000DNAMarker; 1 is the pcr amplification of the COE gene fragment modified without upstream;
Fig. 2 is the Overlap extension PCR amplification of the complete modification fragment of COE gene; M is DL2000DNAMarker; 1 is negative control; 2 is the Overlap extension PCR amplification of the complete modification fragment of COE gene;
Fig. 3 is the PCR qualification result of restructuring E. coli MC1061/pNZ8048-COE; M is DL2000DNAMarker; 1 is negative control; The PCR qualification that 2 is restructuring E. coli MC1061/pNZ8048-COE;
Fig. 4 is the double digestion qualification result of recombinant plasmid pNZ8048-COE; M1 is DL5000DNAMarker; M2 is DL2000DNAMarker; 1 is the double digestion qualification of recombinant plasmid pNZ8048-COE;
Fig. 5 is the PCR qualification result of Recombinant Lactococcus lactis L.lactisNZ9000/pNZ8048-COE; M is DL2000DNAMarker; 1 is negative control; 2 is the PCR qualification of Recombinant Lactococcus lactis L.lactisNZ9000/pNZ8048-COE;
Fig. 6 is the immunoblot results of albumen in Recombinant Lactococcus lactis culture supernatant; 1 is the immunoblot results of the restructuring bacterium culture supernatant without inductor; 2 is the immunoblot results of the restructuring bacterium culture supernatant of 1ng/mL inductor for final concentration; 3 is the immunoblot results of the restructuring bacterium culture supernatant of 2ng/mL inductor for final concentration;
Specific IgA antibody level in Fig. 7 test mice enteron aisle; NS is that saline control group is write a Chinese character in simplified form; The bigeminy Attenuate vaccine group that PEDV+TGEV is commercialization is write a Chinese character in simplified form; NZ9000-COE is writing a Chinese character in simplified form of the Recombinant Lactococcus lactis L.lactisNZ9000/pNZ8048-COE oral vaccine group of this test structure.
Embodiment
Below in conjunction with Figure of description and specific embodiment, set forth the present invention further. These embodiments are only not used in for illustration of the present invention and limit the scope of the invention. The experimental technique of unreceipted concrete condition in lower example embodiment, usually according to this area normal condition or according to the condition of manufacturer's suggestion. Unless otherwise defined, the same meaning that in literary composition, all specialties of using and science term and those skilled in the art are familiar with.
Embodiment 1 is containing the structure of the secretor type Recombinant Lactococcus lactis of COE gene
S1. the structure of secretor type recombinant plasmid pNZ8048-COE
S11. the codon preference optimization of related gene sequence and synthesis: according to the feature of the sequence of goal gene PEDVCOE gene and expression vector pNZ8048, and the signal peptide sequence SP increased for reaching the object of efficient secretory expressionUsp45[(aminoacid sequence is: LEISSTCDA, after on position is Usp45 signal peptide, before goal gene COE, is in centre, and position is closed and is: (PnisA::SP with enhancement sequencesUsp45:: LEISSTCDA::COE]), and for ease of His(Histidine that detection is added) sequence label, to the expressed sequence of whole expection special website (http:// genomes.urv.es/OPTIMIZER) on carry out milk-acid bacteria and express codon preference optimization, adopt the method for synthetic to send company to synthesize the codon optimised sequence of COE gene 420bp. The method of Overlap extension PCR is adopted to carry out the connection of signal peptide and enhancement sequences, design many primers NcoI-Usp1, Usp2, Usp3, Usp4, Usp5, Usp6-COE-F, COE-F, COE-His-HindIII-R, wherein COE-F is the partial dna sequence from COE gene first, carries out to facilitate Overlap extension PCR modifying and design; COE-His-HindIII-R is the downstream primer containing 6 �� His label and HindIII restriction enzyme site, and the 5 ' end at HindIII adds 3 protection base CCC, and 3 ' end adds the terminator codon CTA of reverse complemental, then connects 6 �� His sequence label; NcoI-Usp1 holds the upstream primer of most junior one section sequence containing with the 5 ' of the restriction enzyme site NcoI and signal peptide Usp45 of pNZ8048 amalgamation and expression, NcoI is the amalgamation and expression restriction enzyme site containing initiator codon ATG, adding 4 protection base CATG at its 5 ' end, 3 ' end adds the base AA of two adjustment reading frames; Usp2 ~ 5 are the Overlapping PCR primer of section in the middle of signal peptide; Usp6-COE-F is the primer of signal peptide end section and COE gene initial section stitching portion. Have also been devised primer pNZ1 and pNZ2 of the PCR detection for recombinant plasmid and order-checking simultaneously, it is taking the region of MCS upstream and downstream about the 70��90bp of pNZ8048 empty plasmid as according to designing. The COE gene order of optimum synthesis is as shown in SEQIDNO:1; NcoI-Usp1, Usp2, Usp3, Usp4, Usp5, Usp6-COE-F, COE-F, COE-His-HindIII-R, pNZ1, pNZ2 primer sequence of optimum synthesis is respectively as shown in SEQIDNO:2 ~ 11.
The pcr amplification of the COE gene fragment S12. modified without upstream: taking the plasmid of the COE gene containing optimum synthesis as template, add high-fidelity DNA polymerase PrimeSTARMaxPremix (2 ��) 25 �� L, the each 1 �� L of primer COE-F, COE-His-HindIII-R of 10 ��Ms, template 0.5 �� L, uses ddH2O mends to 50 �� L, and PCR response procedures is: 98 DEG C of denaturation 5min; 98 DEG C of sex change 10s, 55 DEG C of annealing 10s, 72 DEG C extend 5s, 30 circulations; 10min is extended after 72 DEG C. PCR has reacted, product carries out 1.5% sepharose observe and reclaim, visible size is about the amplified band of 450bp, consistent with expected results (such as Fig. 1), and the template as Overlap extension PCR is used for obtaining the complete fragment that with the addition of the modification sequences such as signal peptide by the product of recovery.
The Overlap extension PCR amplification procedure of the complete modification fragment of S13.COE gene: the PCR primer reclaimed in S12 is as template, add high-fidelity DNA polymerase PrimeSTARMaxPremix (2 ��) 25 �� L, the each 1 �� L of primer NcoI-Usp1, Usp2, Usp3, Usp4, Usp5, Usp6-COE-F, COE-His-HindIII-R of 10 ��Ms, template 0.5 �� L, uses ddH2O mends to 50 �� L, and PCR response procedures is: 98 DEG C of denaturation 5min; 98 DEG C of sex change 10s, 55 DEG C of annealing 10s, 72 DEG C extend 5s, 30 circulations; 10min is extended after 72 DEG C. PCR has reacted, and product carries out 1.5% sepharose and observes and reclaim, it is seen that size is about the amplified band of 580bp, consistent with expected results (such as Fig. 2).
S14. the structure of recombinant plasmid pNZ8048-COE: the PCR primer NcoI reclaimed in S13, HindIII are carried out double digestion process, glue reclaims the band that size is about 570bp, in the same way pNZ8048 empty plasmid being carried out double digestion, glue reclaims the band that size is about 3300bp. the pNZ8048 empty plasmid that after getting the complete modification fragment of COE gene and 1 �� L double digestion that glue reclaims after 4 �� L double digestions respectively, glue reclaims, add the efficient ligase enzyme SolutionI of DNALigationKit of 5 �� L, mixed even be placed on 4 DEG C of conditions under connect and spend the night, product Transformed E .coliMC1061 competent cell will be connected, containing 10 �� g/mL paraxin (Chloramphenicol, Cm) in LB agar plate, cultivate two days for 37 DEG C, then choose and get single bacterium colony, it is inoculated in the LB liquid nutrient medium containing 10 �� g/mLCm respectively, more than 3h is cultivated in 37 DEG C of 220rpm joltings, get bacterium liquid and carry out PCR qualification. PCR identifies taking bacterium liquid to be checked as template, adds archaeal dna polymerase 2 �� EsTaqMasterMix10 �� L, and each 0.5 �� L of primer pNZ1, pNZ2, template 0.5 �� L, uses ddH2O mends to 20 �� L, and PCR response procedures is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 45s, 30 circulations; 10min is extended after 72 DEG C. PCR primer detects with 1% agarose gel electrophoresis, it is seen that size is about the amplified band of 730bp, consistent with expected results (such as Fig. 3). The bacterium liquid plasmid DNA extraction agent box that detection is positive is carried out plasmid extraction, and carry out double digestion qualification and sequencing, there is the band of about 570bp and 3300bp two entry in double digestion rear electrophoresis, and check order qualification result and expection consistent (such as Fig. 4), be and obtain recombinant plasmid pNZ8048-COE.
S2. the structure of the secretor type Recombinant Lactococcus lactis of COE gene is contained
S21. the preparation of Lactococcus lactis Electroporation-competent cells: frozen L.lactisNZ9000 Lactococcus lactis is drawn the recovery of G-SGM17 plate, choose and get single bacterium colony 30 DEG C of overnight incubation in G-SGM17 liquid nutrient medium, with the G-SGM17 liquid nutrient medium 30 DEG C cultivation that the ratio of 1:100 access 50mL is new, monitoring OD600To 0.2��0.3, putting rapidly cooled on ice, 4 DEG C of centrifugal 20min of 6000 �� g, abandon supernatant; With the 0.5M sucrose of 50mL precooling, the 10% resuspended thalline of glycerine solution, 4 DEG C of centrifugal 20min of 6000 �� g, abandon supernatant; With the 0.5M sucrose of 25mL precooling, 10% glycerine, the resuspended thalline of 50mMEDTA solution, 4 DEG C of centrifugal 15min of 6000 �� g, abandon supernatant; Again with the 0.5M sucrose of 15mL precooling, the 10% resuspended thalline of glycerine solution, 4 DEG C of centrifugal 15min of 6000 �� g, abandon supernatant; Finally with the 0.5M sucrose of 500 �� L precoolings, the 10% resuspended thalline of glycerine solution, being Lactococcus lactis bacterium competence cell, 40 �� L often pipe packing ,-80 DEG C save backup.
S22. the electric shock conversion of Lactococcus lactis and the PCR qualification of transformant: get 30 �� LL.lactisNZ9000 competent cells, ice bath melts, add 1 �� LpNZ8048-COE recombinant plasmid, mixed even gently; Above mixture is proceeded to the 2mm electric shock cup of ice precooling, give rapidly a monopulse, optimum configurations is 2kV, 25 �� F, 200 ��, softly add the recovery media G-M17MC of 1mL ice precooling immediately, bacterium liquid are all sucked a sterile centrifugation tube after electric shock, cover tightly pipe lid, 30 DEG C of quiescent culture 1��1.5h after ice bath 5min; Bacterium liquid is divided into 10 �� L, 100 �� L, 900 �� L are spread evenly across G-M17C flat board, 30 DEG C of quiescent culture 1��2 day. Choose and get single bacterium colony, it is inoculated in G-M17C liquid nutrient medium respectively, 30 DEG C of more than quiescent culture 5h, get bacterium liquid and carry out PCR qualification, specific operation process is as described in S14, and just template changes Recombinant Lactococcus lactis bacterium liquid to be checked into, and PCR primer detects with 1% agarose gel electrophoresis, the amplified band of visible about 730bp, positive recombinant expressed bacterium called after L.lactisNZ9000/pNZ8048-COE(is such as Fig. 5).
S3. containing the secretor type Recombinant Lactococcus lactis external evoked expression process of COE gene: with the ratio of 1:100, recombinant expressed for L.lactisNZ9000/pNZ8048-COE bacterium is inoculated in G-M17C liquid nutrient medium, and 30 DEG C of quiescent culture spend the night; Overnight culture is inoculated in 10mLG-M17C liquid nutrient medium with the ratio of 1:50, continues to cultivate about 2.5h and enter logarithmic phase (OD to bacterium600=0.4��0.6); It is divided into induction group and does not induce group, it is 1 or 2ng/mL that induction group adds inductor (Nisin) to the final concentration of 1 �� g/mL, terminate cultivating after induction 3h, 4 DEG C of centrifugal 5min of 10000rpm, collect culture supernatant, through SDS-PAGE electrophoresis and carry out WesternBlot analysis, result shows, compared with not inducing group, induction group culture supernatant detects the object band (such as Fig. 6) of 17.38KDa, shows that goal gene has obtained secreting, expressing and had immunocompetence.
Comparative example 1 is containing the structure of the secretor type Recombinant Lactococcus lactis of COE gene
The secretor type Recombinant Lactococcus lactis containing COE gene is built, uniquely the difference is that COE gene used is without codon optimized COE gene fragment with method described in embodiment 1.
Different species have the codon of its preference, this test is according to the result of study of forefathers, have chosen the NZ9000 in L.lactisMG1363 source and express bacterium, and have studied through codon optimized and without constructed by codon optimized COE gene Lactococcus lactis express COE albumen efficiency, find after testing, the COE expressing quantity expressed without the Lactococcus lactis constructed by codon optimized COE gene is lower than 0.001ug/mL, and optimised goal gene COE obtains expression in Lactococcus lactis NZ9000, expression product accounts for the 2.19% of bacterial protein. Concentration reaches 0.62ug/mL.
The application of embodiment 2 Lactococcus lactis in preparation PEDV vaccine
L.lactisNZ9000/pNZ8048-COE Recombinant Lactococcus lactis embodiment 1 prepared makes a kind of oral vaccine preventing porcine epizootic diarrhea, and studies the immune effect of this vaccine, comprises the steps:
The preparation of S1.L.lactisNZ9000/pNZ8048-COE Recombinant Lactococcus lactis oral vaccine: recombinant expressed for L.lactisNZ9000/pNZ8048-COE bacterium is inoculated in G-M17C liquid nutrient medium with the ratio of 1:100, 30 DEG C of quiescent culture spend the night, overnight culture is inoculated in 10mLG-M17 liquid nutrient medium with the ratio of 1:50, continue to cultivate about 2.5h and enter logarithmic phase to bacterium, inductor (Nisin) to the final concentration adding 1 �� g/mL is 2ng/mL, terminate cultivating after induction 6h, full culture now directly uses as oral vaccine, the concentration adopting gradient dilution coated plate method to measure restructuring bacterium reaches 1012The CFU/mL order of magnitude.
S2.L.lactisNZ9000/pNZ8048-COE Recombinant Lactococcus lactis is as the using method of oral vaccine: be the immune effect of preliminary study restructuring bacterium, the female BALB/c mouse in 84 6 week ages is divided at random 3 groups of raisings, often organize 28,1st group is saline control, the 2nd group of immunity full culture suspension (i.e. oral vaccine) of recombinant lactic acid bacteria, the 3rd group of immunity TGEV/PEDV bigeminy Attenuate vaccine. After raising one week in advance, adopting the mode of gavage to carry out oral immunity respectively at the 1st day, the 14th day, each continuous immunity two days, dosage is 160 �� L/. Respectively at immunity before and within after initial immunity the 7th, 14,21,28,35,42,49,56 day, carry out sample collecting, often group gathers 3 mouse every time, comprise periphery blood serum and enteron aisle sample, and 21d, 35d also acquire periphery anticoagulation respectively after first immunisation, it is separated spleen cell, for the detection of different index.
Using COE albumen as ELISA envelope antigen, detection finds, in recombinant lactic acid bacteria group test mice enteron aisle, Specific IgA antibody level reached peak value at the 56th day, higher than other two groups, such as Fig. 7, display L.lactisNZ9000/pNZ8048-COE recombinates bacterium can as the candidate vaccine of induction enteron aisle local mucous membrane immunity. The periphery blood CD8 of 35d+T cell ratio is significantly higher than NS group, and higher than TGEV/PEDV bigeminy Attenuate vaccine group, splenocyte COE stimulates CD8 in lower T lymphocyte+Cell proportion is also the highest; 21d splenocyte COE albumen stimulates the IFN �� concentration in culture supernatant to be significantly higher than NS group, higher than TGEV/PEDV bigeminy Attenuate vaccine group; IFN ��, the IL-6 concentration of 35d are significantly higher than NS group, and higher than TGEV/PEDV bigeminy Attenuate vaccine group, display L.lactisNZ9000/pNZ8048-COE restructuring bacterium also has certain effect for the cellullar immunologic response improving body. These results tentatively confirm that this recombinant lactic acid bacteria has the potentiality as oral vaccine, for development of new oral vaccine prevents PED to provide certain theoretical basis.
SEQUENCELISTING
<110>Agricultural University Of South China
<120>a kind of secreting, expressing PEDV cAg COE Protein reconstitution Lactococcus lactis and its preparation method and application
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Claims (7)
1. the preparation method of a secreting, expressing Porcine epidemic diarrhea virus cAg COE Protein reconstitution Lactococcus lactis, it is characterised in that, comprise the following steps:
S1. the COE gene of synthetic after codon optimized, designs many primers, adopts the method for Overlap extension PCR to be connected on COE gene by Usp45 signal peptide sequence;
S2. the COE gene that S1 obtains is connected to pNZ8048 and screens and obtain recombinant plasmid pNZ8048-COE; Adopt electrotransformation to import in Lactococcus lactis L.lactisNZ9000 by pNZ8048-COE recombinant plasmid, obtain Recombinant Lactococcus lactis L.lactisNZ9000/pNZ8048-COE;
COE gene order after codon optimized described in S1 is as shown in SEQIDNO:1, and each primer sequence is as shown in SEQIDNO:2��9; The primer sequence of described screening recombinant plasmid is as shown in SEQIDNO:10��11.
2. the preparation method of Recombinant Lactococcus lactis according to claim 1, it is characterised in that, the response procedures of Overlap extension PCR described in S1 is: 98 DEG C of denaturation 5min; 98 DEG C of sex change 10s, 55 DEG C of annealing 10s, 72 DEG C extend 5s, 30 circulations; 10min is extended after 72 DEG C.
3. the preparation method of Recombinant Lactococcus lactis according to claim 1, it is characterized in that, electrotransformation described in S2 is: get L.lactisNZ9000 competent cell, add pNZ8048-COE recombinant plasmid, mix even and proceed to electricity and transform cup, recovery media is added, quiescent culture after ice bath, plate screening height copy transformant after electric shock.
4. the recombinant lactic acid bacteria galactococcus L.lactisNZ9000/pNZ8048-COE that the described method of the arbitrary item of claims 1 to 3 prepares.
5. Recombinant Lactococcus lactis L.lactisNZ9000/pNZ8048-COE described in claim 4 prevents the application in porcine epizootic diarrhea oral vaccine in preparation.
6. one kind is prevented the preparation method of porcine epizootic diarrhea oral vaccine, it is characterized in that, comprise the steps: Recombinant Lactococcus lactis L.lactisNZ9000/pNZ8048-COE described in claim 4 is inoculated in G-M17C liquid nutrient medium, quiescent culture spends the night, get overnight culture and it is inoculated in G-M17 liquid nutrient medium with certain proportion, continue to be cultured to bacterium and enter logarithmic phase, adding inductor Nisin to final concentration is 1��2ng/mL, induction certain time, the full culture after induction is directly as oral vaccine.
7. the oral vaccine of the prevention porcine epizootic diarrhea that method described in claim 6 prepares.
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