CN110295134A

CN110295134A - A kind of building and its application of surface display c-type perfringens alpha, β toxin protein recombinant plant lactobacillus

Info

Publication number: CN110295134A
Application number: CN201910546869.9A
Authority: CN
Inventors: 王欣; 李超; 倪宏波
Original assignee: Heilongjiang Bayi Agricultural University
Current assignee: Heilongjiang Bayi Agricultural University
Priority date: 2019-06-24
Filing date: 2019-06-24
Publication date: 2019-10-01

Abstract

The present invention relates to a kind of surface display c-type perfringens alphas, β toxin protein recombinant plant lactobacillus, are the gene orders of 2 toxin of the gene order for being inserted into c-type clostridium perfringens alpha toxin respectively in the genome of lactobacillus plantarum, the gene order of 1 toxin of β and β.The invention further relates to the construction method of above-mentioned recombinant plant lactobacillus and applications.The present invention clones α, β 1 and 2 toxin gene of β by PCR method success from c-type C.perfringens (CVCC 1158) full-length genome; construct corresponding recombinant plasmid; and electricity is transferred in lactobacillus plantarum NC8; after inducing expression; obtain three plant weight group lactobacillus plantarums; select oral way that mouse is immunized; and the immune effect of the recombinant bacterium is evaluated; it can stimulate body generation humoral immunity that body can also be stimulated to generate cellular immunity the result shows that mouse is immunized in oral recombinant plant lactobacillus, have good protective effect to animal body.

Description

A kind of surface display c-type perfringens alpha, β toxin protein recombinant plant lactobacillus Building and its application

Technical field

The invention belongs to Animal molecular biology field, in particular to a kind of surface display c-type perfringens alpha, β poison The building and its application of fibroin recombinant plant lactobacillus.

Background technique

It is that a kind of energy generation is pathogenic outer that C.perfringens (Clostridium perfringen), which is also referred to as clostridieum welchii, The anaerobic spore-bearing bacilli of toxin, there are many exotoxin type caused by C.perfringens, and wherein α, β, ε and ι are main lethal Property toxin, according to the difference for generating lethal toxins, C.perfringens can be divided into five types of A, B, C, D and E, alpha toxin and β Toxin is the principal causative toxin of c-type C.perfringens.C.perfringens infect during alpha toxin be important it is pathogenic because Element, in numerous bacteriotoxins, which, which is first, not only has enzymatic activity, but also again with the bacterium egg of toxin property It is white.β toxin is the principal causative toxin of c-type C.perfringens, by being found in animal experiment, c-type C.perfringens β poison After plain gene is knocked, the mutant strain is caused to lose virulence, the vigor of toxin after wild-type beta toxin gene adds to mutant It will bring back to life, 1 toxin of β is lethal, gangrenosum acne the pore-forming toxins of one kind encoded by cpb1 gene, and 1 toxin of β is thin for endothelium Born of the same parents have virulent property, destroy cell cytoskeleton, cell membrane atrophy so as to cause cell death, 1 toxin of β has the work of dissolution cell Property, it can induce the hemorrhagic necrosis of intestinal mucosa, jejunum is the major lesions position of 1 detoxifying function of β, but 1 toxin of β does not have cell Splitting action.1 toxin of β can influence permeability of cell membranes in conjunction with cell surface receptor, make intraor extracellular ion imbalance, draw Edema and cracking are played, when 1 detoxifying function of β is when central nervous system, affected animal tic of limbs etc. is caused to react.2 toxin of β by Cpb2 gene coding, have with 1 toxin of β faint immune-related, which can cause the gangrenosum acne of piglet Enteritis.

Piglet red dysentery is one kind as caused by c-type C.perfringens (Clostridium perfringen type c) The most common enteric infection disease, the disease cause the disease incidence of grice diarrhoea to reach 40%-50%, and the death rate is up to 100%, And morbidity is anxious, dead fast, and medication effect is bad after the onset, which has report in the farm of China's most area Road causes great economic loss to the pig breeding industry in China, this disease is typically due to a variety of toxin while acting on, toxin intrusion Blood causes enterotoxemia, so should prepare the immunogene of a variety of toxin when preparing vaccine while use, it is most main to prevent the disease The approach wanted is exactly immunity inoculation, and at present when preventing piglet red dysentery, what is mainly utilized is the immune pregnancy of inactivation toxoid vaccine Sow.But traditional inactivated vaccine the disadvantages of there is inactivations to be not thorough, and virulence is anti-strong.Current toxoid vaccine is Be widely used, but since its inactivating substance is formaldehyde, it is bigger to the loss of antigen, be extremely difficult to one it is satisfactory Inactivation condition.

C-type C.perfringens mainly generates 2 three kinds of lethal toxins of α, β 1 and β, and toxin is absorbed by intestinal mucosa infection to be drawn Animal dead is played, so the immune primary stimuli intestinal mucosa for preparing a variety of toxin causes local mucosa-immune to be the pass for preventing the disease Key, lactic acid bacteria are the probiotics in enteron aisle, are played the role of for the stable state of intestinal flora vital.Lactobacillus plantarum conduct One kind of lactic acid bacteria has adjuvant effect, adhesion and the ability for inhibiting most of pathogenic bacteria breedings, utilizes lactobacillus plantarum The oral vaccine that delivery vector as foreign protein develops piglet red dysentery has research significance.

Summary of the invention

The first object of the present invention is to provide a kind of surface display c-type perfringens alpha, β toxin protein recombinant plant Lactobacillus solves c-type perfringens alpha, β 1,2 toxin of β by mucosal infections animal, causes piglet red dysentery problem.

The second object of the present invention is to provide above-mentioned surface display c-type perfringens alpha, β toxin protein recombinant plant The construction method of lactobacillus.

The third object of the present invention is to provide above-mentioned surface display c-type perfringens alpha, β toxin protein recombinant plant The application of lactobacillus.

The fourth object of the present invention is to provide above-mentioned surface display c-type perfringens alpha, β toxin protein recombinant plant The administration mode of lactobacillus.

The present invention is achieved through the following technical solutions:

One, a kind of surface display c-type perfringens alpha, β toxin protein recombinant plant lactobacillus, are in plant cream bar The gene order of c-type clostridium perfringens alpha toxin, 2 toxin of gene order and β of 1 toxin of β are inserted into the genome of bacterium respectively Gene order.

Specifically, the gene order of c-type clostridium perfringens alpha toxin is as shown in SEQ ID No.1, c-type perfringens shuttle The gene order of 1 toxin of bacterium β is as shown in SEQ ID No.2, the gene order such as SEQ ID of 2 toxin of c-type C.perfringens β Shown in No.3.

Two, the construction method of above-mentioned surface display c-type perfringens alpha, β toxin protein recombinant plant lactobacillus, tool Steps are as follows for body:

Step 1: the amplification of c-type perfringens alpha, β 1,2 toxin target gene fragment of β；

Step 2: recombinant plasmid pSIP409- is sieved to obtain in connection, the conversion of PCR product and pSIP409-pgsA ' carrier PgsA '-α, pSIP409-pgsA '-β 1 and pSIP409-pgsA '-β 2；

Step 3: recombinant plasmid pSIP409-pgsA '-α, pSIP409-pgsA '-β 1 and pSIP409-pgsA '-β 2, Electrotransformation lactobacillus plantarum NC8 sieves to obtain NC8-pSIP409-pgsA '-α, NC8-pSIP409-pgsA '-β 1 and NC8- pSIP409-pgsA’-β2。

Specifically, expanding the primer sequence in the step 1 are as follows:

α-F:5'-GGCTCTAGATGGGATGGAAAGATTGATGGAACAGG-3'(SEQ ID No.4)

α-R:5'-GGCAAGCTTTTTTATATTATAAGTTGAATTTCCTGAAATCCACTC-3'(SEQ ID No.5)

β 1-F:5'-GGCTCTAGAATGAAGAAAAAATTTATTTCATTA-3'(SEQ ID No.6)

β 1-R:5'-CGGGGTACCCTAAATAG CTGTTACTTTGTGAGT-3'(SEQ ID No.7)

β 2-F:5'-GGCTCTAGAATGAAAAAAATTATTTCAAA-3'(SEQ ID No.8)

β 2-R:5'-CGGGGTACCCTATGCACAATACCCTTCAC-3'(SEQ ID No.9)。

Three, above-mentioned surface display c-type perfringens alpha, β toxin protein recombinant plant lactobacillus are in anti-perfringens shuttle Application in bacterium α, β 1,2 toxin of β.

Four, above-mentioned surface display c-type perfringens alpha, β toxin protein recombinant plant lactobacillus administration mode be glutinous Film administration.

Specifically, the mucosal drug delivery is oral.

Specifically, the mucous membrane of the surface display c-type perfringens alpha, β toxin protein recombinant plant lactobacillus is given Medicine Effective Antigens amount ratio is NC8-pSIP409-pgsA '-α: NC8--pSIP409-pgsA '-β 1:NC8-pSIP409- PgsA '-β 2=1:1:1.

Good effect by adopting the above technical scheme: the present invention is successful from c-type C.perfringens by PCR method α, β 1 and 2 toxin gene of β are cloned on (CVCC 1158) full-length genome, construct pSIP409-pgsA '-α, pSIP409- 2 plasmid of pgsA '-β 1 and pSIP409-pgsA '-β, and electricity is transferred in lactobacillus plantarum NC8, after inducing expression, obtains three Plant weight group lactobacillus plantarum NC8-pSIP409-pgsA '-α, NC8-pSIP409-pgsA '-β 1 and NC8-pSIP409- PgsA '-β 2 selects oral way that mouse is immunized, and evaluates the immune effect of the recombinant plant lactobacillus, the results showed that Mouse, which is immunized, in oral recombinant plant lactobacillus can stimulate body generation humoral immunity that body can also be stimulated to generate cellular immunity, right Animal body has good protective effect, albumen and the pgsA ' egg as surface display original part of the expression of recombinant plant lactobacillus White amalgamation and expression can solve the problems, such as the extracellular transhipment of recombinant plant lactobacillus expression foreign protein；The successful table of destination protein It up in phage surface, can be come into full contact with intestinal mucosa, stimulation intestinal mucosa causes local mucosa-immune；Three plant weight groups of building are planted Object lactobacillus expresses three kinds of toxin proteins respectively, and enteron aisle is reached after oral, and it is red to prevent piglet for panimmunity primary stimuli intestinal mucosa The effect of dysentery is more preferable, lays the foundation for further research and development C.perfringens oral vaccine；Lactobacillus plantarum is as lactic acid bacteria One kind having adjuvant effect, adhesion and the ability for inhibiting most of pathogenic bacteria breedings, using lactobacillus plantarum as external source The oral vaccine that the delivery vector of albumen develops piglet red dysentery can produce more preferably mucosa-immune effect.

Detailed description of the invention

Fig. 1 is toxin gene PCR products electrophoresis map of the present invention；

Fig. 2 is the physical map of pSIP409-pgsA ' of the present invention；

Fig. 3 is recombinant plasmid double digestion qualification figure of the present invention；

Fig. 4 is the PCR qualification result of recombinant plant lactobacillus of the present invention；

Fig. 5 is the Western-blot detection that alpha toxin albumen of the present invention is expressed in recombinant plant lactobacillus；

Fig. 6 is the Western-blot detection that 1 toxin protein of β of the present invention is expressed in recombinant plant lactobacillus；

Fig. 7 is the Western-blot detection that 2 toxin protein of β of the present invention is expressed in recombinant plant lactobacillus；

Fig. 8 is recombinant plant lactobacillus indirect immunofluorescence result (1000 ×) of the present invention；

Fig. 9 is Bacteria Culture of the present invention and Gram's staining result；

Figure 10 is that the present invention slightly proposes ectotoxic SDS-PAGE testing result；

Figure 11 is c-type C.perfringens chromatography map of the present invention；

Figure 12 is the variation tendency of IgG antibody in each group immune serum of the present invention；

Figure 13 is that sIgA antibody ELISA testing result (OD450nm value) in stool in mice is immunized in each group of the present invention；

Figure 14 is the variation tendency that sIgA antibody in mouse salivary is immunized in each group of the present invention；

Figure 15 is the testing result of IFN-γ of the present invention；

Figure 16 is the testing result of IL-4 of the present invention；

Figure 17 is percentage of the CD4+ cell subsets in total lymphocyte in the immune mouse spleen of the present invention；

Figure 18 is percentage of the CD8+ cell subsets in total lymphocyte in the immune mouse spleen of the present invention.

Specific embodiment

C-type C.perfringens (CVCC 1158): it is protected purchased from China, China Veterinery Drug Inspection Office veterinary microorganism strain Hide administrative center；

Lactobacillus plantarum NC8 and plasmid pSIP409-pgsA ': the recombinant plant cream based on pgsA as surface display element The building and verifying of bacillus expression system, Cai Ruopeng, Master's thesis, conferring unit: Jilin Agriculture University, tutor: Wang Chunfeng, hair The table time 2015.

Embodiment 1

Embodiment 1 illustrates surface display c-type perfringens alpha, the building side of β toxin protein recombinant plant lactobacillus Method:

1, the amplification of target gene:

According to three kinds of toxin gene sequences of c-type C.perfringens on GenBank (M24904.1, KP064405.1, L77965.1), in conjunction with the actual conditions design primer of expressed sequence and carrier.

α-F:5'-GGCTCTAGATGGGATGGAAAGATTGATGGAACAGG-3'

α-R:5'-GGCAAGCTTTTTTATATTATAAGTTGAATTTCCTGAAATCCACTC-3'

β 1-F:5'-GGCTCTAGAATGAAGAAAAAATTTATTTCATTA-3'

β 1-R:5'-CGGGGTACCCTAAATAG CTGTTACTTTGTGAGT-3'

β 2-F:5'-GGCTCTAGAATGAAAAAAATTATTTCAAA-3'

β 2-R:5'-CGGGGTACCCTATGCACAATACCCTTCAC-3'

Primer is synthesized by Harbin Xin Hai genetic test Co., Ltd, introduces Xba I, Hind respectively at the end primer 5' I restriction endonuclease sites of III and Kpn, underscore mark restriction enzyme site.

Using c-type C.perfringens genome as template, with designed primer amplification target gene, PCR system such as table 1 It is shown:

Table 1PCR system additive amount

Upstream primer (20mM)	1μL
		Downstream primer (20mM)	1μL
C-type C.perfringens genome	1μL
		DNTP mixture (2.5mM)	2μL
MgCl2	2μL
		DNA polymerase buffer	2.5μL
Aqua sterilisa	14.5μL
		Taq DNA polymerase	1μL

PCR reaction condition: 1.94 DEG C of initial denaturation 5min；2.94 DEG C of thermal denaturation 30s, 55.3 DEG C of annealing 60s, 72 DEG C extend 1min is recycled 35 times；3.72 DEG C of extension 10min, toxin gene PCR products electrophoresis map are as shown in Figure 1, in which: M:DNA marker DL2 000；1: negative control；2: alpha toxin gene magnification result；3: β 1 toxin gene amplification；4: β 2 toxin base Gene-amplification result.

2, the connection of PCR product and pSIP409-pgsA ' carrier:

Three kinds of pcr amplification products are subjected to 0.8% agarose gel electrophoresis, recycle purpose piece using plastic recovery kit Section, operating procedure refer to Tiangeng plain agar sugar gel DNA QIAquick Gel Extraction Kit (DP 209) explanation in Chinese book, use restriction enzyme Enzyme Xba I and Hind III utilizes double digestion method to alpha toxin gene PCR glue recovery product, then will be at its digestion identical as process PSIP409-pgsA ' carrier after reason；With restriction enzyme Xba I and Kpn I using double digestion method to 1 toxin gene of β and β 2 toxin gene PCR glue recovery products carry out digestion, then by its digestion identical as process treated pSIP409-pgsA ' load Body.After 37 DEG C of water-bath digestion 6h, then digestion products are recycled with DNA purification kit, the physics of pSIP409-pgsA ' For map as shown in Fig. 2, being attached after purification, linked system is as follows: pSIP409-pgsA ' carrier: 1 μ L；T4DNA ligase: 1 μL；10 × T4DNA ligase buffer:1 μ L；PCR product: the total volume of 7 μ L, reaction are 10 μ L, avoid acutely rocking, be put in In 16 DEG C of connection instrument, 8-12h is connected.

3, it converts:

The connection product of 10 μ L is slowly added into 100 μ L TOP10 competent cells, is stood after being placed in 30min on ice It is put into 42 DEG C of water-baths and places 90s, place 5min on ice immediately after.600 μ are added into converted product after terminating for ice bath L LB liquid medium is placed in 37 DEG C of shaking tables, and 180r/min cultivates 1h, and the bacterium solution 5000r/min for cultivating 1h is centrifuged 3min discards 600 μ L supernatants, is spread evenly across the LB solid containing 100ug/mL erythromycin after having been hanged precipitating with remaining liq On culture medium, 37 DEG C of incubator culture 12h.

4, the identification of recombinant plasmid:

4 bacterium colonies on three LB plates of picking are inoculated in the LB Liquid Culture that 5mL contains 100ug/mL erythromycin respectively In base, overnight incubation in 37 DEG C of shaking tables extracts plasmid, and operating procedure is with reference to the small extraction reagent kit of Tiangeng plasmid (DP103-02) Chinese Specification.According to the restriction endonuclease of the restriction enzyme site selection double digestion effect on the target gene of amplification, the plasmid of extraction is carried out Digestion products are carried out 0.8% agarose gel electrophoresis, double digestion are identified that correct recombinant plasmid is sent to Kazakhstan by double digestion identification The measurement that bodyguard biology Co., Ltd carries out sequence is won in your shore, and three kinds of correct plasmids of sequencing are named as pSIP409-pgsA '-α, PSIP409-pgsA '-β 1 and pSIP409-pgsA '-β 2, recombinant plasmid double digestion qualification figure is as shown in figure 3, result proves PSIP409-pgsA '-α plasmid 6167bp and 1 plasmid of 1110bp, pSIP409-pgsA '-β 6173bp and 1011bp, There is purpose band at 6173bp and 798bp in 2 plasmid of pSIP409-pgsA '-β, it was demonstrated that three kinds of recombinant plasmids successfully construct, weight It is compared after group plasmid order-checking through BLAST, homology reaches 99% or more.Wherein, M:DNA marker DL10 000；1, again Group plasmid pSIP409-pgsA '-α double digestion identification；2,1 double digestion of recombinant plasmid pSIP409-pgsA '-β is identified；3, matter is recombinated Grain 2 double digestion of pSIP409-pgsA '-β identification.

5, the preparation of lactobacillus plantarum NC8 competence:

Lactobacillus plantarum NC8 strain is taken out in ultra low temperature freezer, is inoculated in 5mL MRS culture solution, is subsequently placed at and detests After cultivating 12h in oxygen incubator, three rides connect bacterium on MRS plate, and 30 DEG C of incubator overnight incubations are chosen single bacterium colony, taken 5mL contains the MRS fluid nutrient medium of 2% glycine, is subsequently placed into anaerobic jar and continues culture to bacterium solution OD₆₀₀=0.6 or so； Then secondary culture, then the single bacterium colony of picking are inoculated in 5mL MRS fluid nutrient medium (2% glycine), as bacterium solution OD₆₀₀= When 0.4 or so, bacterium solution is placed in 20min on ice, bacterium solution is utilized into low temperature ultracentrifuge, by thallus centrifugation, collects bacterium Body precipitating.Bacterium is resuspended with buffer, low-temperature centrifugation cleans twice.Precipitating is resuspended with electroporation buffer, is in charge of spare.

6, the electrotransformation of recombinant plasmid:

Take recombinant plasmid the pSIP409-pgsA '-α, pSIP409-pgsA '-β 1 and pSIP409-pgsA '-β built 2 each 3 μ L are separately added into 100 μ l lactobacillus plantarum NC8 competence, get out the electric shock cup of pre-cooling in advance, by recombinant plasmid and Competent cell mixture is placed in 10min on ice after the cup mixing that shocks by electricity, and parameter (2.5kV, 6ms) is arranged and carries out electricity turn； After electricity turns, by the cup ice bath 5min that shocks by electricity, in the EP pipe for the 2ml for being added to 850 μ L MRS culture solutions into electric shock cup, 37 DEG C of anaerobism Under the conditions of cultivate 2.5-3h, containing Em (final concentration of 10ng/mL) MRS solid medium on the 200 above-mentioned bacterium of μ L of even spread Liquid is cultivated under 37 DEG C of anaerobic conditions to growing single bacterium colony in good condition.Picking single bacterium colony carries out pure culture, is incubated overnight Recombinant plant lactobacillus DNA is extracted afterwards, carries out PCR identification.

According to pgsA ' gene in recombinant plant lactobacillus, specific primer is designed:

UP 5'-CAACAAAAAAAGAAGAC-3'(SEQ ID No.10)

LOW 5'-TAATGAATAACCAGCAC-3'(SEQ ID No.11)。

Amplification obtains the target fragment of about 480bp from the recombination after electrotransformation, as a result as shown in figure 4, result proves structure The three kinds of plasmids built successfully are transferred in three lactobacillus plantarum NC8.Wherein M:DNA marker DL2 000；1:NC8- PSIP409-pgsA '-α amplification；1 amplification of 2:NC8-pSIP409-pgsA '-β；3:NC8-pSIP409-pgsA '-β 2 Gene magnification result；4: negative control.

Embodiment 2

Embodiment 2 illustrates that recombinant plant lactobacillus is identified:

1, the destination protein concentration of BCA method measurement expression:

The three plant weight group lactobacillus plantarum inducing expressions that will be built, and ultrasonication is carried out, concrete operation step reference Green skies BCA protein concentration detection kit specification, each group recombinant plant lactobacillus express the concentration of albumen, as a result such as table 2:

2 BCA of table detects protein concentration

Recombinant plant lactobacillus group	Protein concentration (mg/ml)
		pSIP409-pgsA’-α	0.11148
pSIP409-pgsA’-β1	0.21048
		pSIP409-pgsA’-β2	0.21106
pSIP409-pgsA’	0.42870

2, Western-blot is detected:

In order to whether be inferred to express albumen in phage surface expression, protein sample is handled using two methods, ultrasound is broken Broken method and multigelation method, by comparing expression and the combination indirect immunofluorescence of albumen as a result, determining the table of albumen Up to position.Protein sample is subjected to electrophoresis, transferring film.After transferring film, pvdf membrane is placed in room temperature in the confining liquid just prepared It is gentle to shake closing 1.5h, 3 each 10min are washed with TBST.The polyclonal antibody of preparation is diluted with TBST according to 1:200, Pvdf membrane after transferring film is placed in the serum of preparation, 4 DEG C are incubated overnight；Pvdf membrane after washing is placed in use In mountain sheep anti-mouse igg antibody antibody of the TBST by the diluted horseradish enzyme of 1:2 000 label, 1h, TBST washing 3 are incubated in 37 DEG C of shaking tables It is secondary；Finally developed the color using DAB colour reagent box, photograph to obtain as a result, experimental result as illustrated in figs. 5-7, experimental result It is high-visible to show that pSIP409-pgsA '-α group sample occurs at 61kDa after sonioation method and the processing of multigelation method Protein band；1 group of sample of pSIP409-pgsA '-β occurs at 52kDa after sonioation method and the processing of multigelation method High-visible protein band；2 groups of samples of pSIP409-pgsA '-β sonioation method and multigelation method processing after Occurs high-visible protein band at 46kDa.Wherein M in Fig. 5: low molecule quality egg is from matter standard；1: empty carrier The expression of pSIP409-pgsA '；2: sonioation method；3: multigelation method；M in Fig. 6: low molecule quality egg is from matter standard；1: Sonioation method；2: multigelation method；3: the expression of empty carrier pSIP409-pgsA '；M in Fig. 7: low molecule quality egg is from matter mark It is quasi-；1: sonioation method；2: multigelation method；3: the table of empty carrier pSIP409-pgsA '.

3, indirect immunofluorescence is identified

2ml is taken to induce the recombinant plant lactobacillus of 5h, 12000r/min is centrifuged 1min, and 0.5% BSA is contained with 1ml PBS wash 3 times, collect precipitating, precipitating be put into more anti-(1:200 dilution) room temperature shakers incubations that preparation is added in 6 orifice plates 1h；It is washed 3 times with the PBS containing 0.2%Tween 20, collects precipitating, sheep anti mouse FITC antibody (dilution of 1:2 000) room is added It under the conditions of temperature, is placed in shaking table and is incubated for 45min, 12 000r/min of bacterium solution of collection is centrifuged 1min, is discarded supernatant, precipitating is with containing There is the PBS of 0.2%Tween 20 to wash 3 times, collect precipitating, after having been hanged with 200 μ L PBS, 2 μ l of absorption are evenly coated in adherency and carry On slide, fluorescence microscope result is as shown in figure 8, through fluorescence microscope as it can be seen that control group phage surface does not find green Fluorescence, three groups of test group can be found that the higher rod-short thallus of fluorescence intensity, in conjunction with Western-blot as a result, proving expression Three kinds of c-type clostridium perfringens toxoid protein expressions on the surface of the thallus of recombinant plant lactobacillus plantarum.Wherein in figure from It is left-to-right to be followed successively by empty carrier pSIP409-pgsA ' group；PSIP409-pgsA '-α group；1 group of pSIP409-pgsA '-β； 2 groups of pSIP409-pgsA '-β.

Embodiment 3

Embodiment 3 expresses the Efficacy evaluation of perfringens alpha, β toxin protein recombinant plant lactobacillus:

1, animal immune is grouped

Female BAl BIc/c the mouse for taking 150 5 week old, is divided into 3 groups, separately raises, marks.It will build simultaneously The recombinant plant lactobacillus 5000r/min of overnight induction is centrifuged 6min, and the PBS of bacterial sediment sterilizing is cleaned, and cleans three repeatedly After, viable count is calculated, every each immunity inoculation viable count is 1 × 10⁹cfu.On day 1, it the 3rd day, carries out within the 5th day first Immune, the 20th day, the 22nd day, the 24th day progress booster immunization are wherein prohibited before oral immunity and raise 4~6h, during which guarantee sufficient drink Water is taken a blood sample weekly, acquires excrement and saliva carries out index of correlation detection.Specific immunization protocol such as the following table 3:

Animal packet is immunized in table 3

Group	Vaccine	Immunizing dose (200 μ L)	Immunization route	Size of animal
					1	PBS group	200μL	It is oral	50
2	Lactobacillus plantarum empty carrier group (pSIP409-pgsA ')	1×10⁹cfu	It is oral	50
					3	Recombinant plant lactobacillus (experimental group)	1×10⁹cfu	It is oral	50

Note: experimental group NC8-pSIP409-pgsA '-α, NC8--pSIP409-pgsA '-β 1 and NC8-pSIP409- 2 mixed in equal amounts of pgsA '-β.

2, ectotoxic preparation

By the c-type C.perfringens normal saline dilution of freeze-drying, sterile working is inoculated into certainly in anaerobic culture box In sheep blood plate processed, 37 DEG C of cultures carry out gram stain microscopy for 24 hours, to bacterium, as a result as shown in figure 9, on sheep blood plate Grow in good condition, surface is smooth, neat in edge and have double zone of hemolysis bacterium colony c-type C.perfringens, to it Gram's staining is carried out, dyes visible single or multiple Gram-positives under mirror, coarse bacillus, after dyeing microscopic examination is qualified, picking Single bacterium colony is inoculated in 100ml meat liver gastric enzyme digest medium, is incubated overnight in 37 DEG C of anaerobic box, and 5ml overnight incubation is drawn Bacterium solution be inoculated in 100ml meat liver gastric enzyme digest medium again, 43 DEG C of Anaerobic culturel 6h, by 12 000r/ of gained bacterium solution Min centrifugation 10min takes supernatant, and obtains c-type perfringens shuttle exotoxin liquid with 0.22 μm of membrane filtration.

3, ectotoxic slightly to mention

(1) saturated ammonium sulfate solution is added slowly to it is in the exotoxin solution of above-mentioned preparation and stirring while adding, Make sulfuric acid by concentration reach 50%, shake up 4 DEG C and stand overnight；

(2) 10 000r/min of mixed liquor is centrifuged 20min, abandons supernatant, precipitating is molten with the Tris-HCL buffer of 0.05M Solution；

(3) saturated ammonium sulfate solution is added, when final concentration reaches 40%, shakes up 4 DEG C of standings and places 8-12h；

(4) 10 000r/min of mixed liquor is centrifuged 20min, abandons supernatant, precipitating will be dissolved with Tris-HCl buffer；

(5) it to suitable sample is added in processed super filter tube before the test, is put after super filter tube is balanced Enter centrifuge；

(6) centrifuge is adjusted to slow and is accelerated, low temperature ultracentrifuge 3000r/min is centrifuged 10min, and mixed liquor can divide Repeatedly it is added；

(7) after the completion of all samples are concentrated, desalination Buffer is added, centrifugal ultrafiltration after balance, twice in succession；

(8) it operates on ice and uses liquid-transfering gun gentle aspiration recovery product；

(9) after the completion of by the super filter tube made according to cleaning is illustrated, 20% ethyl alcohol will be filled in super filter tube, 4 DEG C of preservations are standby With.

The c-type C.perfringens of preparation is slightly mentioned into exotoxin sample, takes 10 μ L to carry out polyacrylamide gel electrophoresis, such as Shown in Figure 10, the results showed that have obvious band at 43KD, 34KD and 28KD, meet 2 toxin protein size of toxin α, β 1 and β.

One, oral immunity mouse detection of specific antibody

1, molecular sieve gel chromatography the preparation of envelope antigen: is carried out to the toxin slightly mentioned using Biologic Chromatography system:

(1) the verified exotoxin sample that slightly mentions containing toxin protein will be obtained and uses 0.22 μm of filter membrane mistake before chromatography Filter selects Mini Bio-P-6Cartridges 5mL chromatographic column；

(2) it extracts bubble in A and B pump out, so that there is no bubble appearance in pump, tightens interface, first start A after starting software Pump, the 1L ultrapure water rinse of use sterilized degassing process；

(3) the PBS buffer solution cleaning of reasonable employment sterilizing and balance pillar, the flow velocity of 5mL/min is balanced, when ultraviolet With conductance baseline it is steady after start to lose shape；

(4) when the curve risen occurs in absorbance, start to collect chromatographic solution, use 50%BaCl during collection₂Solution prison Survey the SO of every pipe collection liquid₄ ^2-, when there is precipitating to generate, stop collecting immediately, it is right after the chromatographic solution collected using super filter tube concentration The protein concentration of concentrate is detected.

Chromatography map is as shown in figure 11, collects the chromatographic solution in peak value according to tomographic map, during collecting chromatographic solution, In order to reduce SO₄ ^2-Influence to later period test, adds 50%BaCl to chromatographic solution in the process₂Solution, detection toxin concentration are 3.13mg/mL。

2, in mice serum specific IgG antibodies detection

For acquisition 200 μ L of blood in 1.5mL EP pipe, centrifugal process separates blood after mouse is docked weekly before immune and after immune Clearly, and antibody test is carried out, detailed step refers to Heilongjiang Bayi Agricultural Reclamation University Master's thesis-Liu Zenglu-A, C, D type gas for ox The Preliminary development and Efficacy evaluation of capsular clostridium trivalent toxoid vaccine, negative control group serum is acquired in advance.

Ig G antibody test result is as shown in Figure 12 and table 4 in ELISA detection mice serum:

IgG antibody ELISA testing result (OD450nm value) in 4 mice serum of table

Note: marking not identical expression significant difference with column data, the not identical expression significant difference (P < 0.05) of lowercase, Capitalization difference indicates that difference is extremely significant (P < 0.01).

The result shows that specificity Ig G antibody level is high compared with empty carrier group and control group in test group serum, although unloaded Body group is more slightly higher than serum specificity Ig G antibody in control group, but the two there is no difference, test group to exempt from two after first week and It is extremely significant (P ﹤ 0.01) that second week antibody level reaches higher level difference compared with other two groups, from two exempt from after exempt to two within two weeks Three weeks rapid decreases afterwards, two to exempt from downward trend after three weeks more steady, illustrates within this period, the recombinant plant lactobacillus of building It has been colonized on intestinal mucosa and has expressed corresponding antigens.

3, in stool in mice and saliva specificity sIgA antibody detection

(1) acquisition and processing of excrement: excrement is put into the EDTA-Na containing 0.05mol/L₂PBS excrement extracting solution in (0.1g excrement is added in every 500 μ L excrement extracting solution) is placed in 4 DEG C of refrigerator 12h, then 12 000r/min of sample is centrifuged 10min, Supernatant is collected, detailed step refers to Heilongjiang Bayi Agricultural Reclamation University Master's thesis-Liu Zenglu-A, C, D type Clostridium perfringens from bovines The Preliminary development and Efficacy evaluation of trivalent toxoid vaccine.

SIg A antibody test result is as shown in Figure 13 and table 5 in ELISA detection stool in mice:

SIgA antibody ELISA testing result (OD450nm value) in 5 stool in mice of table

The result shows that test group mouse generates specificity sIg by feeding recombinant plant lactobacillus energy effective stimulus body A, empty carrier group are more slightly higher than sIg A antibody in the stool in mice of control group, but the two there is no difference, test group to exempt from one after Exempt from extremely significant (P ﹤ 0.01) with other two groups of differences in first week, antibody level rapid increase after being immunized every time within one week and two, but It is that duration is poor, lasts about one week greatly, antibody level rapid decrease.

(2) it the acquisition and processing of saliva: is rotated several times, is put into containing 200 μ in the oral cavity of mouse with sterile cotton swab In the 1.5mL EP pipe of L PBS and 20 μ g/mL Aprotinins, after 12 000r/min are centrifuged 5min at room temperature, it are transferred into and go out In the dry 2mL EP pipe of bacterium.

SIg A antibody test result is as shown in Figure 14 and table 6 in ELISA detection mouse salivary:

SIgA antibody ELISA testing result (OD450nm value) in 6 mouse salivary of table

The result shows that latter all test groups are immunized every time than specificity sIg A antibody level in other two groups of mouse salivaries High and difference is extremely significant (P ﹤ 0.01), and immune second week antibody level is declined every time, and test group is compared with other two groups of mouse Specificity sIg A antibody level height and significant difference (P ﹤ 0.05) in saliva, two exempt from each group after three weeks, and there was no significant difference.

It is shown by the above result of indirect ELISA, the recombinant plant lactobacillus of building not only induces local immune response, together When stimulate body generation system humoral immune response.

Two, the detection of Cytokine of Serum

IL-4 is one of the most important factor for influencing Th2 cell differentiation, and IL-4 can effectively facilitate the proliferation of B cell, lure It leads it and generates Ig G antibody.Thl cell Major Secretory IFN-γ, IL-2 and TNF-α, IFN-γ mainly by the T cell that activates and NK cell generates, and is the potential activity factor of mononuclear macrophage, can significantly increase MHC2 class developed by molecule, promotes complementary The activation of T lymphocyte, to enhance the cellullar immunologic response of animal body.

1, in serum IFN-γ detection: according to IFN-γ cytokine detection kits operating instruction to one exempt from after two The content detection that week exempts from IFN-γ in rear two weeks mice serums with two, concrete operation step are shown in R&D Mouse IFN-γ Quantikine ELISA Kit explanation in Chinese book.

IFN-γ assay result is as shown in Figure 15 and table 7 in mice serum:

IFN-γ content in 7 immune serum of table

Group	One exempts from two weeks afterwards	Two exempt from two weeks afterwards
			Test group	63.47±4.18Aa	71.27±4.52Aa
Empty carrier group	55.30±3.30b	61.16±3.27b
			Control group	50.58±3.29Bb	50.90±5.12Cc

The result shows that test group and empty carrier group, two contents for exempting to exempt from rear two weeks IFN-γ for latter two weeks than one have obviously Raising, one exempt from after two weeks, test group is extremely significant compared with control group difference (P ﹤ 0.01), test group compared with empty carrier group difference show It writes (P ﹤ 0.05), but empty carrier group and control group difference is not significant, two exempt from two weeks afterwards, and difference is extremely aobvious compared with the control group for test group It writes (P ﹤ 0.01), test group significant difference (P ﹤ 0.05) compared with empty carrier group, empty carrier group significant difference compared with the control group (P ﹤ 0.05) illustrates that the content of IFN-γ in test group mice serum is all remarkably higher than other two groups, to enhance mouse body Cellullar immunologic response.

2, in serum interleukin-4 (IL-4) detection: according to the operating instruction of IL-4 cytokine detection kits Exempt from latter two weeks and two to exempt from the content detection of IL-4 in rear two weeks mice serums to one, concrete operation step is shown in R&D Mouse IL-4Quantikine ELISA Kit explanation in Chinese book.

IFN-γ assay result is as shown in Figure 16 and table 8 in mice serum:

IL-4 content in 8 immune serum of table

Group	One exempts from two weeks afterwards	Two exempt from two weeks afterwards
			Test group	123.26±4.02a	140.95±3.93A
Empty carrier group	117.34±7.07a	124.16±4.32Bb
			Control group	108.97±6.35b	111.04±5.44Bc

Note: marking not identical expression significant difference with column data, the not identical expression significant difference (P < 0.05) of lowercase, Capitalization difference indicates that difference is extremely significant (P < 0.01)

Test group and empty carrier group, two contents for exempting to exempt from rear two weeks IL-4 for latter two weeks than one have raising.One exempts from two weeks afterwards, Test group significant difference (P ﹤ 0.05) compared with the control group, difference is not significant compared with empty carrier group, empty carrier group and control group Significant difference, two exempt from after two weeks, test group difference compared with empty carrier group and control group is extremely significant (P ﹤ 0.01), empty carrier group and Control group compares significant difference (P ﹤ 0.05), illustrates that lactobacillus plantarum up-regulation IL-4 is horizontal, this and Ig G in each group mice serum Antibody level is substantially consistent.

In summary it is small to illustrate that the recombinant plant lactobacillus that builds of feeding can be enhanced for IL-4 and IFN-γ testing result Mouse humoral immunity and cellular immunity.

Three, in spleen CD4+ and CD8+T lymphocyte detection

5 small white mouses of every group of execution after being immunized two weeks every time take spleen to separate lymphocyte, detect CD4 after being immunized⁺With CD8⁺The variation of T lymphocyte, detailed experimental steps refer to Heilungkiang Aug. 1st university master thesis-Chang Chunlong-ox Pasteur The preparation of bacillus bacterium shadow vaccine and its immune efficacy analysis.

1, CD4+T cell subsets accounts for the dynamic change of total lymphocyte percentage in spleen, as shown in Figure 17 and table 9:

Percentage of the CD4+ cell subsets in total lymphocyte in mouse spleen is immunized in table 9

Group	One exempts from two weeks afterwards	Two exempt from two weeks afterwards
			Test group	28.71±0.49a	29.61±0.78a
Empty carrier group	26.58±0.65b	27.19±0.79b
			Control group	26.50±0.54b	26.35±0.53b

CD4 in the mouse spleen of test group and empty carrier group after immune⁺T cell subgroup account for total lymphocyte percentage with it is right According to group compared in rising trend, after exempting from one two weeks and two exempt from after it is equal with empty carrier group and control group difference between two weeks test groups Significantly (P ﹤ 0.05), empty carrier group and control group difference is not significant.

2, CD8+T cell subsets accounts for the dynamic change of total lymphocyte percentage in spleen, as shown in Figure 18 and table 10:

CD8 in immune rear test group and empty carrier group mouse spleen⁺T cell subgroup account for total lymphocyte percentage with compare Group is compared to increased, and two weeks after exempting from one, test group and control group significant difference (P ﹤ 0.05), two exempt between two weeks test groups afterwards With control group significant difference (P ﹤ 0.05), empty carrier group after second immune with control group significant difference (P ﹤ 0.05).

Percentage of the CD8+ cell subsets in total lymphocyte in mouse spleen is immunized in table 10

Group	One exempts from two weeks afterwards	Two exempt from two weeks afterwards
			Test group	17.82±0.25a	18.59±0.61a
Empty carrier group	17.24±0.61b	17.95±0.38ab
			Control group	16.72±0.42b	16.42±0.69c

CD4⁺And CD8⁺It is the surface markers of T lymphocyte, CD4⁺Mainly express in T helper cell to mediate body Based on liquid is immune, CD8⁺Mainly it is expressed in cytotoxic T cell, mediating cellular immune.Two exempt from two phases after rear two Zhou Yuyi exempt from Than CD4 in mouse spleen⁺The percentage that T cell subgroup accounts for total lymphocyte has raising, and test group is poor compared with other two groups It is different significant.CD8 in test group mouse spleen⁺The percentage that T cell subgroup accounts for total lymphocyte has raising, and two exempt from sky after a week Vehicle group and control group significant difference illustrate that lactobacillus plantarum itself can stimulate body to break up CD8⁺T cell, stimulation body produce Raw nospecific immunity.

Embodiment 4

Embodiment 4 illustrates that mouse attacks malicious protectiveness test

One, the measurement of minimum lethal dose:

By obtained c-type perfringens shuttle exotoxin liquid 1mL physiological saline by 2^-5、2^-6、2^-7、2^-8Gradient doubling dilution, 10 mouse of each gradient, every mouse peritoneal inject 500 μ L toxin dilutions, and every mouse peritoneal of control group injects 500 μ L Physiological saline, 72h observe and record dead mouse situation, calculate minimum lethal dose (MLD), the results are shown in Table shown in 11, as a result table It is bright: minimum lethal dose 2^-7/ ml toxin crude extract.

The measurement experiment of 11 minimal lethal dose of table

Group	Toxin extension rate	The death rate (dead sum/sum)
			1	2^-5	100% (10/10)
2	2^-6	50% (5/10)
			3	2^-7	10% (1/10)
4	2^-8	0% (0/10)

Two, challenge test:

After second immune 14d, 4 small white mouses of each immune group are carried out with 3,4 and 5 times of MLD dosage respectively Poison is attacked in intraperitoneal injection, is attacked the clinical symptoms and The dead quantity of record small white mouse after poison, be the results are shown in Table 12:

The experiment of 12 Immunization of table

Group	3MLD	4MLD	5MLD
				Test group	0	2	4
Empty carrier group	4	4	4
				Control group	4	4	4

Note: number is to attack dead mouse number after poison in table.

As the result is shown: test group can be fully against the attack of 3MLD toxin, and when with 4MLD toxin attacks, protective rate is 50%, other test mices are dead in 24 hours.

Three, the detection of mouse neutralizing antibody is immunized:

The sterile test group mice serum for taking two to exempt from latter two weeks carries out toxin neutralization test, with the C of 4 times of minimum lethal doses Type C.perfringens exotoxin and 1:2,1:4,1:8,1:16 times of diluted mice serum mixed in equal amounts, are put in 37 DEG C of incubators and incubate 1h, 0.5mL/ intraperitoneal injection of mice are educated, each extension rate injects mouse 4, observes 24 hours, records mouse survival feelings Condition, as a result as shown in table 11:

1h, 4 mouse of every group of intraperitoneal injection are acted in 37 DEG C of incubators under aseptic condition.The results show that test group mouse blood When clear dilution is 1:4, in toxin and efficiency is 100%.

11 toxin neutralization test of table

Serum dilution	The death rate (dead sum/sum)
		1:2	0% (0/4)
1:4	0% (0/4)
		1:8	25% (1/4)
1:16	100% (4/4)

It attacks malicious protective effect and neutralizes experiment, the test group for feeding lactobacillus plantarum as the result is shown can be fully against 3MLD The attack of toxin, when the attack of 4MLD toxin, protective rate 50%, serum antibody neutralization effect experimental result is shown, test When group mice serum dilution is 1:4, in toxin and efficiency is 100%.

In conclusion this success of the test is prepared for three plant weight group lactobacillus plantarums, NC8-pSIP409-pgsA '-α, NC8- PSIP409-pgsA '-β 1 and NC8-pSIP409-pgsA '-β 2, three plant weight group lactobacillus plantarum mixed in equal amounts of preparation are oral to be exempted from Epidemic disease mouse can induce it to generate stronger humoral immunity and cellular immunity, and good immunoprotection can be provided for mouse.

Sequence table

<110>Heilongjiang Bayi Agricultural Reclamation University

<120>building and its application of a kind of surface display c-type perfringens alpha, β toxin protein recombinant plant lactobacillus

<130> 201901

<141> 2019-06-24

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1110

<212> DNA

<213>C.perfringens (Clostridium perfringens)

<400> 1

tgggatggaa agattgatgg aacaggaact catgctatga ttgtaactca aggggtttca 60

atcttagaaa atgatctgtc caaaaatgaa ccagaaagtg taagaaaaaa cttagagatt 120

ttaaaagaga acatgcatga gcttcaatta ggttctactt atccagatta tgataagaat 180

gcatatgatc tatatcaaga tcatttctgg gatcctgata cagataataa tttctcaaag 240

gataatagtt ggtatttagc ttattctata cctgacacag gggaatcaca aataagaaaa 300

ttttcagcat tagctagata tgaatggcaa agaggaaact ataaacaagc tacattctat 360

cttggagagg ctatgcacta ttttggagat atagatactc catatcatcc tgctaatgtt 420

actgccgttg atagcgcagg acatgttaag tttgagactt ttgcagagga aagaaaagaa 480

cagtataaaa taaacacagc aggttgcaaa actaatgagg ctttttatac tgatatctta 540

aaaaacaaag attttaatgc atggtcaaaa gaatatgcaa gaggttttgc taaaacagga 600

aaatcaatat actatagtca tgctagcatg agtcatagtt gggatgattg ggattatgca 660

gcaaaggtaa ctttagctaa ctctcaaaaa ggaacagcgg gatatattta tagattctta 720

cacgatgtat cagagggtaa tgatccatca gttggaaaga atgtaaaaga actagtagct 780

tacatatcaa ctagtggtga gaaagatgct ggaacagatg actacatgta ttttggaatc 840

aaaacaaagg atggaaaaac tcaagaatgg gaaatggaca acccaggaaa tgattttatg 900

actggaagta aagacactta tactttcaaa ttaaaagatg aaaatctaaa aattgatgat 960

atacaaaata tgtggattag aaaaagaaaa tatacagcat tctcagatgc ttataagcca 1020

gaaaacataa agataatagc aaatggaaaa gttgtagtgg acaaagatat aaacgagtgg 1080

atttcaggaa attcaactta taatataaaa 1110

<210> 2

<211> 1011

<212> DNA

<213>C.perfringens (Clostridium perfringens)

<400> 2

atgaagaaaa aatttatttc attagttata gttagttcac ttttaaacgg atgcctatta 60

tcaccaactt tagtgtatgc aaatgatata ggtaaaacta ctactataac tagaaataag 120

acatcagatg gctatactat aattacacaa aatgataaac agataatatc atatcaatct 180

gttgactctt caagtaaaaa tgaagatggt tttactgcat ctatagatgc tagatttatc 240

gatgataaat attcatctga aatgacaact ttaataaact taactggatt tatgtcttca 300

aaaaaagaag atgttataaa aaaatacaat ttgcatgatg ttactaattc tactgcaatt 360

aattttccgg ttagatactc gatttctatt ttaaatgaaa gtattaatga aaatgtaaaa 420

atagttgata gtattcctaa aaatacaatt tctcaaaaaa ctgtatccaa tacaatggga 480

tacaaaatag gaggttcaat tgaaatagaa gaaaataaac ctaaagcttc aattgaaagc 540

gaatatgctg aatcatctac aatagaatat gtccaacctg atttttctac tatacagaca 600

gatcattcaa cctctaaagc ttcatgggat acaaaattta cagaaactac tcgtggtaat 660

tataatttaa aatcaaacaa ccctgtatat ggaaatgaaa tgtttatgta cggaagatat 720

actaatgttc ctgcaactga aaatataatt ccagattatc aaatgtcaaa attaataaca 780

ggtggtttaa accctaatat gtctgtagtt ctaactgctc ctaatggtac tgaagaatct 840

ataataaaag ttaaaatgga gcgtgaaaga aactgttatt atcttaattg gaatggtgct 900

aactgggtag gacaagtcta ttccaggcta gcttttgata ccccaaatgt agatagtcat 960

atatttacat tcaaaataaa ttggcttact cacaaagtaa cagctattta g 1011

<210> 3

<211> 798

<212> DNA

<213>C.perfringens (Clostridium perfringens)

<400> 3

atgaaaaaaa ttatttcaaa gtttactgta atttttatgt tttcatgttt tcttattgtt 60

ggagcaataa gtccaatgaa agcaagtgca aaagaaatcg acgcttatag aaaggtaatg 120

gagaattatc ttaatgcttt aaaaaactac gatattaata cagttgtaaa catttcagaa 180

gatgaaagag taaataatgt tgaacagtat agagaaatgt tagaagattt taaatatgat 240

cctaaccaac aactgaaatc ttttgaaata cttaattcac aaaagagcga taataaagaa 300

atatttaatg taaaaactga atttttaaat ggtgcaattt atgatatgga atttactgta 360

tcatctaaag atggaaaatt aatagtatct gatatggaaa gaacaaaagt tgagaatgaa 420

ggaaaatata ttttaacacc atcatttaga actcaagttt gtacatggga tgatgaacta 480

gcacaagcaa ttgggggagt ttatccacaa acatattctg atagatttac atattatgca 540

gataatatat tattaaactt cagacaatat gcaacttcag gttcaagaga tttaaaagta 600

gaatatagtg ttgtagatca ttggatgtgg aaagatgatg ttaaagcttc tcaaatggta 660

tatggtcaaa atcctgattc tgctagacaa ataagattat atatagaaaa aggacaatct 720

ttctataaat atagaataag aattaaaaac tttacacctg catcaattag agtatttggt 780

gaagggtatt gtgcatag 798

<210> 4

<211> 35

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

ggctctagat gggatggaaa gattgatgga acagg 35

<210> 5

<211> 45

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

ggcaagcttt tttatattat aagttgaatt tcctgaaatc cactc 45

<210> 6

<211> 33

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

ggctctagaa tgaagaaaaa atttatttca tta 33

<210> 7

<211> 33

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

cggggtaccc taaatagctg ttactttgtg agt 33

<210> 8

<211> 29

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

ggctctagaa tgaaaaaaat tatttcaaa 29

<210> 9

<211> 29

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

cggggtaccc tatgcacaat acccttcac 29

<210> 10

<211> 17

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

caacaaaaaa agaagac 17

<210> 11

<211> 17

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

taatgaataa ccagcac 17

Claims

1. a kind of surface display c-type perfringens alpha, β toxin protein recombinant plant lactobacillus, it is characterised in that: be to plant The gene order of c-type clostridium perfringens alpha toxin, the gene order and β 2 of 1 toxin of β are inserted into the genome of object lactobacillus respectively The gene order of toxin.

2. surface display c-type perfringens alpha according to claim 1, β toxin protein recombinant plant lactobacillus, Be characterized in that: the gene order of c-type clostridium perfringens alpha toxin is as shown in SEQ ID No.1,1 poison of c-type C.perfringens β The gene order of element is as shown in SEQ ID No.2, the gene order of 2 toxin of c-type C.perfringens β such as SEQ ID No.3 institute Show.

3. a kind of surface display c-type perfringens alpha according to claim 1, β toxin protein recombinant plant lactobacillus Construction method, it is characterised in that: specific step is as follows:

Step 2: recombinant plasmid pSIP409-pgsA '-α is sieved to obtain in connection, the conversion of PCR product and pSIP409-pgsA ' carrier, PSIP409-pgsA '-β 1 and pSIP409-pgsA '-β 2；

Step 3: recombinant plasmid pSIP409-pgsA '-α, pSIP409-pgsA '-β 1 and pSIP409-pgsA '-β 2, electricity turn Change lactobacillus plantarum NC8, sieves to obtain NC8-pSIP409-pgsA '-α, NC8-pSIP409-pgsA '-β 1 and NC8-pSIP409- pgsA’-β2。

4. construction method according to claim 3, it is characterised in that: expand the primer sequence in step 1 are as follows:

α-F:5'-GGCTCTAGATGGGATGGAAAGATTGATGGAACAGG-3'(SEQ ID No.4)

α-R:5'-GGCAAGCTTTTTTATATTATAAGTTGAATTTCCTGAAATCCACTC-3'(SEQ ID No.5)

β 1-F:5'-GGCTCTAGAATGAAGAAAAAATTTATTTCATTA-3'(SEQ ID No.6)

β 1-R:5'-CGGGGTACCCTAAATAG CTGTTACTTTGTGAGT-3'(SEQ ID No.7)

β 2-F:5'-GGCTCTAGAATGAAAAAAATTATTTCAAA-3'(SEQ ID No.8)

β 2-R:5'-CGGGGTACCCTATGCACAATACCCTTCAC-3'(SEQ ID No.9)。

5. surface display c-type perfringens alpha described in claim 1, β toxin protein recombinant plant lactobacillus are in anti-production gas Application in capsular clostridium α, β 1,2 toxin of β.

6. surface display c-type perfringens alpha described in claim 1, β toxin protein recombinant plant lactobacillus give prescription Formula is mucosal drug delivery.

7. surface display c-type perfringens alpha according to claim 6, β toxin protein recombinant plant lactobacillus, Be characterized in that: the mucosal drug delivery is oral.

8. surface display c-type perfringens alpha according to claim 6, β toxin protein recombinant plant lactobacillus is viscous It is NC8-pSIP409-pgsA '-α: NC8--pSIP409-pgsA '-β 1:NC8-pSIP409- that Effective Antigens amount ratio, which is administered, in film PgsA '-β 2=1:1:1.