CN102277373B - Schistosoma japonica vaccine expression vector for attenuated salmonella secretion expression and use thereof - Google Patents
Schistosoma japonica vaccine expression vector for attenuated salmonella secretion expression and use thereof Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology and particularly relates to a schistosoma japonica vaccine salmonella secretion expression vector, which is a recombinant salmonella expression vector that is controlled by a bacterial promoter, guided by effector protein secreted by salmonella and secretion signal of the effector protein and used for secretion expression of schistosoma japonica antigen. The schistosoma japonica vaccine salmonella secretion expression vector can induce secretion expression of a schistosoma japonica vaccine in a hypoxic microenvironment in antigen presenting cells, can exist in vivo stably and continuously and is insusceptible to being lost. The schistosoma japonica vaccine salmonella secretion expression vector, by using an attenuated strain as a carrier, can perform the antigen carrying of the recombinant vaccine representing bacteria by means of oral taking. The schistosoma japonica vaccine salmonella secretion expression vector can be used for preparing schistosoma japonica vaccine for oral taking for preventing and treating schistosomiasis.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of Japanese blood fluke vaccine expression vector and application thereof of attenuation salmonella secreting, expressing.
Background technology
Schistosomicide is the infecting both domestic animals and human parasitosis of a kind of serious harm mankind and animal health, and the whole world approximately 600,000,000 people live in schistosomicide epidemic-stricken area, and 200,000,000 people are infected, and clinical symptom appears in 1.2 hundred million people, and year death toll is nearly hundreds thousand of.After working out specifics praziquantel, by colony's chemotherapy, to popular existing alleviation of schistosomicide, but the propagation of schistosomicide is still continuing, and number of patients is still increasing.And the resistance of the chemicals such as praziquantel has also affected the control (McManus DP, Clin Microbiol Rev, 2008,21 (1): 225-242) of schistosomicide to a great extent.Therefore,, for the propagation of better prevention and cure of schistosomiasis and popular, need to develop in a hurry a kind of efficient, safe, inexpensive blood fluke vaccine.
Blood fluke vaccine research has successively been experienced the stages such as killed vaccine, deactivation or attenuated vaccine, recombiant vaccine and nucleic acid vaccine.Although inactivated vaccine has shown higher protection, its risk is high; It is not high that killed vaccine, recombiant vaccine and nucleic acid vaccine are but faced with protection, involve great expense, and inoculation inconvenience etc. problem.Oral antigen submission system has inoculation simply because of it, and low-cost advantage is widely used in the exploitation of multi-infection disease vaccine.The Salmonellas of active attenuation is a kind of desirable potential carrier for oral vaccine delivery, it can pass through oral route, exogenous antigen molecule is transported in antigen presenting cell (APC), and passing through histocompatibility complex's submission to T cell, excitating organism produces cell, body fluid and the mucosal immune response for antigen molecule.
Khan CM attempts utilizing attenuated Salmonell SL3261 to express Schistosoma mansoni 28kd GST antigen first, after oral immunity mouse, antibody (the Khan CM for GST antigen in serum, detected, Proc Natl Acad Sci. 1994,91 (23): 11261-11265); Pacheco LG etc. utilizes attenuation salmonella SL3261 to express Schistosoma mansoni 14 kd lipid acid result albumen, has obtained 34.9% worm reduction rate (Acta Trop. 2005,95 (2): 132-142) after oral mouse.Once but Salmonellas enters after antigen presenting cell, just be limited in a kind of vesica (Salmonella containing vacuole of film parcel, SCV) (Zhang XL etc., Cell Mol Immunol. 2008,5 (2): 91-97) among.The imitated vesicle structure of this film parcel has limited effective submission of antigen to a great extent.Therefore, in order to break this restriction, develop a kind of blood fluke vaccine taking oral attenuation salmonella as carrier efficiently, need to face the secretion problem of antigen molecule.
Therefore, set up an efficient attenuation salmonella secreting, expressing Schistosoma japonicum oral vaccine system, must build an efficient Salmonellas secretion expression carrier, to realize the secreting, expressing of antigen molecule in Salmonellas, this is the problem to be solved in the present invention.
Summary of the invention
The problem that the present invention need to solve is to set up an efficient attenuation salmonella secreting, expressing Schistosoma japonicum oral vaccine system, builds a kind of Salmonellas secretion expression carrier efficiently, to realize the secreting, expressing of antigen molecule in Salmonellas.
Main purpose of the present invention is to build efficient Salmonellas secretion expression carrier; realize antigen molecule and stablize in vivo lasting secreting, expressing, lay the foundation for setting up the Schistosoma japonicum oral vaccine taking attenuation salmonella as transport agent that expression is stable, protectiveness efficiency is high.
In order to achieve the above object, the present invention utilizes III type excretory system and hemolysin excretory system to secrete schistosome antigen molecule, III type secretion signal and hemolysin secretion signal and antigen molecule are merged to realize the secretion to the antigen including Schistosoma japonicum, and described III type secretion signal can be the secretory signal sequence of the protokaryon bacterium III type secretion effect proteins such as Salmonellas outer membrane protein E N end 1-104 aminoacid sequence; Described hemolysin secretion signal is 34 amino acid of hlyA albumen n end and 61 aminoacid sequences of C end; Described antigen molecule is the vaccine antigens such as sj23LHD-GST bivalent antigen.
Further, the invention provides a kind of schistosomicide oral vaccine submission system, it is characterized in that taking attenuation salmonella as oral vaccine delivery vehicles, with the mode submission Japan schistosome antigen molecule of secreting, expressing, described attenuated bacteria strain can be attenuation salmonella VNP20009.
Further, the invention provides a kind of stable Schistosoma japonicum secreting, expressing oral vaccine taking attenuation salmonella as carrier, plasmid is expressed stable in vivo, does not lose.
Compared with existing blood fluke vaccine, characteristics and innovation part of the present invention is:
(1) invent one and utilized Salmonellas secreting, expressing signals direct Japanese blood fluke vaccine antigen expression vector, realize the secreting, expressing of Schistosoma japonicum bivalent antigen, and confirm to utilize this system at mouse model, can realize liver worm reduction rate and reach 41.69%, liver egg reduction rate is 57.71%.
(2) invent a kind of Japanese blood fluke vaccine of salmonella-mediating of oral form, and can obtain the higher humoral immunization for antigen molecule and cell immune response at oral this vaccine of the confirmations such as animal infection modal.
(3) invented a kind of Japanese blood fluke vaccine with low cost, the present invention is owing to having invented the Japanese blood fluke vaccine antigen expression vector of a kind of high efficient expression, efficient secretion, add and utilize the convenient Salmonellas of cultivating as antigen presentation factory and submission instrument, there is the application prospect of large-scale promotion.
In sum, the invention provides a kind of brand-new, efficient, safe, cheap, easily, oral, can be used for Japanese blood fluke vaccine secretion expression carrier and the application thereof of humans and animals.
Four, brief description of the drawings
The structure of figure mono-, attenuation salmonella secretion expression carrier
(1A) large hydrophilic area and the gst fusion protein of schistosome antigen sj23LHDGST(sj23 molecule) the structure schematic diagram of secretion expression carrier.Two kinds of excretory systems are built.1, III type secreting, expressing system; 2, hemolysin secreting, expressing system.PnirB and phlyA are respectively nitrite reductase promotor and hemolysin gene A(hlyA) promotor; SopE1-104 is Salmonellas III type excretory system secretion signal; HlyA is hemolysin secretion signal, and hlyB, hlyC, hlyD are the required element of hemolysin excretory system.
(1B) the recombinant salmonella secreting, expressing antigen molecule of Western bolting checking secreting, expressing is to the situation in nutrient solution supernatant.1, unloaded bacterium; 2, restructuring hemolysin secretion bacterium; 3, restructuring III type secretion bacterium is normally cultivated; 4, restructuring III type secretion bacterium anaerobism is cultivated.The nirB promoter expression antigen that III type secretion bacterium is regulated and controled by anaerobism, it is expressed needs weary oxygen environment to induce.
(1C) recombinant salmonella of secreting, expressing infects after antigen presenting cell (mouse macrophage RAW264.7), the expression by analogue antigen EGFP submission to scavenger cell.DAPI dyeing represents nucleus, 1, unloaded bacterium; 2, restructuring III type secretion bacterium; 3, restructuring hemolysin secretion bacterium.
Recombinant attenuated Salmonellas blood fluke vaccine titre and the time length in vivo of figure bis-, secreting, expressing
(2A) BALB/C mice oral 10
9after the recombinant vaccine strain of colony-forming unit (cfu) secreting, expressing and wild mushroom in 3-20 days, the titre of recombinant bacterium in peripheral immune organ spleen.1, wild mushroom (non-transformed Salmonellas), represents total count; 2, restructuring hemolysin secretion bacterium; 3, restructuring III type secretion bacterium.The recombinant salmonella of secreting, expressing sustainable existence in spleen, vaccine plasmid is not lost.
(2B) BALB/C mice oral 10
9after the recombinant salmonella of cfu secreting, expressing and wild mushroom in 3-20 days, the titre of recombinant bacterium in peripheral immune organ lymphoglandula.1, wild mushroom (non-transformed Salmonellas), represents total count; 2, restructuring hemolysin secretion bacterium; 3, restructuring III type secretion bacterium.The recombinant salmonella of secreting, expressing sustainable existence in lymphoglandula, vaccine plasmid is not lost.
The immune response for antigen molecule sj23LHDGST that after the recombiant vaccine bacterium of figure tri-, BALB/C mice oral vaccination secreting, expressing, body produces.
(3A) the oral unloaded bacterium of mouse, restructuring III type secretion bacterium and restructuring hemolysin are secreted bacterium three times, and serum biweekly, is got in after last immunity one week in interval, the IgG antibody titer of anti-sj23LHDGST antigen in serum analysis.1, unloaded bacterium; 2, restructuring III type secretion bacterium; 3, restructuring hemolysin secretion bacterium.
(3B) oral immunization in mice PBS, unloaded bacterium, restructuring hemolysin secretion bacterium and restructuring III type secretion bacterium (10
9cfu) three times, spleen biweekly, is got in after last immunity one week in interval, after erythrocyte splitting, prepares splenocyte suspension, then uses respectively recombinant antigen sj23LHD, GST, and positive control ConA stimulates 72 hours, analyzes splenocyte secretion of gamma-IFN amount.1, PBS; 2, unloaded bacterium; 3, restructuring hemolysin secretion bacterium; 4, restructuring III type secretion bacterium.
Restructuring blood fluke vaccine bacterium postabdomen subcutaneous infection Schistosoma japonicum protectiveness efficiency analysis after 42 days of figure tetra-, oral immunization in mice secreting, expressing.
(4A) BALB/C mice oral immunity PBS, unloaded bacterium, restructuring hemolysin secretion bacterium and restructuring III type secretion bacterium (10
9cfu) three times, biweekly, after last immunity one week, subcutaneous abdomen infected 40 of schistosoma japonicum cercariaes at interval, infected to calculate for latter 42 days in Mice Body, to become borer population.1, PBS; 2, unloaded bacterium; 3, restructuring hemolysin secretion bacterium; 4, restructuring III type secretion bacterium.
(4B) one-tenth borer population × 100% of worm reduction rate=(the average every mouse of one-tenth borer population-experimental group of the average every mouse of control group becomes borer population)/average every mouse of control group.1, unloaded bacterium; 2, restructuring hemolysin secretion bacterium; 3, restructuring III type secretion bacterium.
(4C) method of utilizing 5% potassium hydroxide (KOH) digestion liver to spend the night, calculates liver worm's ovum number.1, PBS; 2, unloaded bacterium; 3, restructuring hemolysin secretion bacterium; 4, restructuring III type secretion bacterium.
(4D) egg reduction rate=(the average every mouse liver worm's ovum number of the average every mouse liver worm's ovum number-experimental group of control group) average every mouse liver worm's ovum number × 100% of/control group.1, unloaded bacterium; 2, restructuring hemolysin secretion bacterium; 3, restructuring III type secretion bacterium.
Five, embodiment:
Taking large hydrophilic area and the gst fusion protein of Schistosoma japonicum bivalent antigen sj23LHDGST(sj23 molecule) as delivering antigen as embodiment:
1, the structure of the vaccine carrier of secreting, expressing:
Antigen molecule sj23LHD-GST adopts respectively Salmonellas III type excretory system and hemolysin excretory system to secrete.
A) structure of III type secretion expression carrier: by 1-104 amino acid (sopE of III type secretion effector molecule sopE N end
1-104) realize its secretion with antigen molecule sj23LHD coupling; Adopt intestinal bacteria nitrate reductases (nirB) promotor to express III type secretion antigen.NirB promotor utilizes PCR method to obtain: primer sequence is: P1:5 '-cccctcgagggttaccggcccgatcg-3 '; P2:5 '-cccggatccaccgcctaccttaacgattc-3 '; With 50 ng genome of E.coli DNA as template.The fragment two ends that obtain are with Xho I and BamH I restriction enzyme site, and PCR program is all 94 DEG C of 40 s, 60 DEG C of 40 s, 72 DEG C of 1 min, 26 circulations.Salmonellas III type excretory system secretion signal is the amplification of sopE albumen 1-104 aminoacid sequence gene: primer sequence is: P1:5 '-cccggatccatgactaacataacactatc-3 '; P2:5 '-aaaggtacccggatctttactcgcat-3 '; The sopE1-104 gene two ends that obtain are respectively with the restriction enzyme site of BamH I and Kpn I, and PCR program is all 94 DEG C of 40 s, 60 DEG C of 40 s, 72 DEG C of 1 min, 26 circulations.The acquisition of bivalent vaccine gene sj23LHDGST: adopt overlapping PCR method amplification to obtain sj23LHDGST gene, primer sequence is: P1:5 '-aaaggtaccatgtacaaggataaaatcgatg-3 '; P2:5 '-taagttgcgttttaagaatgctagtataggggacat-3 '; P3:5 '-ttcttaaaacgcaacttaatgtcccctatactaggt-3 '; P4:5 '-cccaagcttttattttggaggatggtcgc-3 '; There is method as follows: first respectively with P1 and P2, P3 and P4 are primer taking pcDNA3.1-sj23 and pcDNA3.1-GST as template, amplify sj23LHD fragment and GST fragment; Then taking P1 and P4 as primer, taking the sj23LHD fragment expanded and GST fragment as template obtains sj23LHDGST bivalent vaccine gene, the sj23LHDGST gene two ends of increasing are respectively with Kpn I and Hind III restriction enzyme site.PCR program is all 94 DEG C of 40 s, 60 DEG C of 40 s, 72 DEG C of 1 min, 26 circulations.The connection of promotor-secretion signal-antigen gene: select low copy expression vector pQE30, first nirB promotor is connected between the Xho I and BamH I of pQE30 carrier; Secondly by secretion signal sopE
1-104insert between BamH I and Kpn I site; Then bivalent vaccine antigen gene sj23LHDGST gene is inserted between Kpn I and Hind III site.After transforming, identify, checking order, obtain III type secretion expression carrier nirB-sopE
1-104-sj23LHDGST.The enzyme program of cutting is wherein: 20 microlitre reaction systems, 37 DEG C, 3 hours; Linker is: 10 microlitre reaction systems, room temperature, 5 hours.
B) hemolysin excretory system is delivered the structure of vaccine antigen carrier: utilize hemolysin secretion expression carrier pMohly1 to realize the hemolysin secreting, expressing of antigen, this carrier contains the necessary all elements of hemolysin excretory system and secretion signal, and adopts the original promotor of effector molecule hlyA to express.Sj23LHDGST gene is connected in the Nsi I of pMohly1 single endonuclease digestion site, and consistent with upper and lower reading frame; Primer sequence for the sj23LHDGST gene that increases is: P1:5 '-ccaatgcatcgtacaaggataaaatcg-3 '; P2:5 '-ccaatgcatcttttggaggatggtcgc-3 '; Amplification PCR program is 94 DEG C of 40 s, 60 DEG C of 40 s, 72 1 26 of min circulations.Then amplified production and carrier pMohly1 are carried out to Nsi I enzyme and cut, endonuclease reaction system 20 microlitres, 37 DEG C, 3 hours; After enzyme is cut product and reclaimed, carrier carries out dephosphorization acid with alkaline phosphatase, and reaction system is 20 microlitres, and 37 DEG C, 3 hours; Finally carrier is connected with fragment, reaction system is 10 microlitres, room temperature 5 hours.After transforming, identify, checking order, obtain hemolysin secretion vector pMohly1-sj23LHDGST.
2, the electroporation of recombinant attenuated Salmonellas transforms:
Salmonellas electricity turns competent preparation: inoculate fresh attenuation salmonella in 200 ml LB substratum, 37 DEG C of shaking tables are cultured to OD value between 0.4-0.6, centrifugal 6000g × 5 min collects thalline, with aseptic double-distilled water washing thalline one time, centrifugal 6000 g × 5 min, with the washing thalline of 10% glycerine two times, centrifugal 6000 g × 5 min, resuspended with 500 microlitre 10% glycerine, packing 50 microlitres/pipe, turns for electricity.
Adopt electroporation method that recombiant vaccine DNA vector is transformed in attenuation salmonella VNP20009: under aseptic condition, the recombinant vectors (secretion of III type or hemolysin secretion) that 0.5-5 μ g is built is added in electricity and turns in competence, after mixing, transfer in the electric revolving cup of 0.2 μ M, for electric shock, it is 1.8 KV that electricity turns condition, 500 Ω, 2 μ F, electricity turns rear painting ammonia benzyl plate screening, and the bacterium colony growing is recombinant bacterium.
3, immune programme for children and method:
6-8 female mice BALB/C in age in week divides into groups according to 20 every group, every mouse oral 1 × 10
9the unloaded bacterium group of cfu bacterium dose inoculation, restructuring III type secretion group, restructuring hemolysin secretion group, the oral 0.2 ml PBS of natural control group.Oral immunity three times, interval biweekly, is carried out schistosomicide for one week for the third time afterwards.
4, recombiant vaccine bacterium titre in vivo detects:
BALB/C mice oral 10
9cfu restructuring III type secretion bacterium, restructuring hemolysin secretion bacterium and the wild attenuation salmonella not transforming, got respectively spleen and mesenteric lymph nodes at 3 days, 10 days, 20 days, weigh, according to 5:1(PBS volume: Organ weight) ratio add PBS to carry out homogenate, then homogenate is carried out to gradient dilution, be applied to ammonia benzyl flat board, according to the titre of recombinant bacterium in longer colony number and Organ weight calculating internal organs, non-transformed wild attenuation salmonella is coated with the LB flat board that normally there is no resistance, calculates total count with this.
5, sj23LHDGST antibody test:
The preparation of immune serum: BALB/C mice is according to above-mentioned immune programme for children inoculated bacteria vaccine, after last immunity one week, tail vein blood, room temperature left standstill after 2-4 hour, and centrifugal 5 minutes of 5000 rpm collect serum;
Sj23LHDGST antigen coated: utilize 50 mM, the carbonate buffer solution of pH9.6 is according to 10 μ g/ml dilution antigens, and every hole drips 100 μ l, and 4 degree are coated to spend the night, PBST wash after three times (5 minutes once) for detection of.
ELISA measures antibody titers: the every hole of coated plate adopts the BSA of 200 μ l 3% 37 degree sealing 3 hours, wash (5 minutes once) three times through PBST, then use 100 μ l PBS by serum gradient dilution 50 extraordinarily in hand-hole, 37 DEG C are reacted 1-2 hour, PBST washes (5 minutes once) three times, adding HRP(horseradish peroxidase) goat anti-mouse igg of coupling two is anti-, 37 DEG C are reacted 1 hour, PBST washes (5 minutes once) three times, add tmb substrate colour developing, the 2 M vitriol oils stop, and measure 450 nM absorb light.
6, IFN-γ detects:
Last immunity is after one week, get three mouse spleens for every group, under aseptic condition, make single cell suspension, after erythrocyte splitting, adjusting cell concn with the RPMI 1640 of 10% foetal calf serum is 2 × 106/ml, joins in 96 orifice plates according to every hole 100 microlitres, stimulates 72 hours respectively with 10 ug/ml sj23LHD, 10 ug/ml GST, 10 ug/ml Con A, collecting cell culture supernatant, adopt IFN-γ ELISA test kit, to specifications, measure IFN-γ content
7, immanoprotection action is observed:
After last immunity one week, every mouse infected 40 ± 2 schistosoma japonicum cercariaes through skin of abdomen, after infecting, within 6 weeks, cutd open and killed, and collects adult calculate worm lotus mean by perfusion; Take liver 0.5 g, add 10 ml5% KOH, after 37 DEG C of digestion 5 h, get 0.1 ml smear, counting under the microscope, i.e. every gram of hepatic tissue worm's ovum number (EPG), evaluates each group of plasmid immune protective efficiency with worm reduction rate and every gram of liver egg reduction rate.Calculation formula is as follows: worm reduction rate (%)=[(control group worm lotus mean-experimental group worm lotus mean)/control group worm lotus mean] × 100%; Egg reduction rate (%)=[(control group EPG-experimental group EPG)/control group EPG] × 100%.
1) and hemolysin excretory system (Figure 1A: 2) the attenuation salmonella VNP20009 of embodiment of the present invention utilization safety is carrier, adopts two kinds of excretory systems to carry out secreting, expressing to Schistosoma japonicum bivalent antigen sj23LHD-GST: III type excretory system (Figure 1A:.Utilize the method that electricity transforms to transform after attenuation salmonella, the III type that obtains recombinating is secreted bacterium, restructuring hemolysin secretion bacterium and is transformed the unloaded bacterium of unloaded plasmid, the secretion situation of each recombinant bacterium to antigen that adopted western bloting technology for detection, result is presented in the nutrient solution supernatant of unloaded bacterium and sj23LHD-GST antigen (Figure 1B: 1) do not detected; Restructuring hemolysin secretion bacteria culture fluid supernatant (Figure 1B: 2) and restructuring III type secrete bacterium anaerobism culture supernatant in (Figure 1B: 4) antigen all can be detected; Because what III type excretory system adopted is the nirB promotor of weary oxygen regulation and control, therefore in restructuring III type secretion bacterium aerobic culture supernatant, antigen (Figure 1B: 3) do not detected.Carry situation in order further to prove the antigen of recombinant bacterium in antigen presenting cell-scavenger cell, the present invention adopts EGFP(green fluorescent protein) as analogue antigen, infect in vitro after macrophage system RAW264.7 when carrying each recombinant bacterium of EGFP, infect after unloaded bacterium, in scavenger cell born of the same parents, do not observe fluorescence (Fig. 1 C:1); Infect after restructuring III type secretion bacterium (Fig. 1 C:2) and restructuring hemolysin secretion bacterium (Fig. 1 C:3), fluorescence in scavenger cell born of the same parents, all can be detected.
In order to evaluate the plasmid stability of each recombinant bacterium, each recombinant bacterium and wildness are unconverted bacterium is according to 1 × 10
9cfu/ oral dose is only inoculated the 5-7 Balb/C mouse in age in week.Within 3,10,20 days after inoculation, separate peripheral immune organ spleen and lymphoglandula, calculate the titre of each recombinant bacterium in organ.In spleen, with respect to wild mushroom (Fig. 2 A:1), restructuring hemolysin secretion bacterium (Fig. 2 A:2) and restructuring III type secretion bacterium (Fig. 2 A:3) all reduce less than obvious in each time point titre; In lymphoglandula, be also like this equally, with respect to wild mushroom (Fig. 2 B:1), restructuring hemolysin secretion bacterium (Fig. 2 B:2) and restructuring III type secretion bacterium (Fig. 2 B:3) all reduce less than obvious in each time point titre.
The present invention has further evaluated the immune response for antigen molecule producing after each oral each recombiant vaccine bacterium.Oral immunization in mice PBS, unloaded bacterium and recombiant vaccine bacterium three times, interval biweekly, is got serum for after last immunity one week, and the antibody of evaluating anti-sj23LHD-GST in serum produces.With respect to unloaded bacterium (Fig. 3 A:1), restructuring III type secretion bacterium (Fig. 3 A:2) and restructuring hemolysin secretion bacterium (Fig. 3 A:3) can produce the antibody special for antigen molecule, and the III of wherein recombinating type secretion bacterium (Fig. 3 A:2) antibody titer is the highest.Because the oral vaccine taking Salmonellas as carrier is mainly induced body generation Th1 reaction, so the present invention is also respectively with restructuring sj23LHD, restructuring GST, and positive control medicine Con A immune stimulatory mouse boosting cell, analyze the generation of splenocyte secrete cytokines IFN-γ, compare with unloaded bacterium (Fig. 3 B:2) immunity with PBS immunity (Fig. 3 B:1), restructuring hemolysin secretion bacterium (Fig. 3 B:3) and restructuring III type secretion bacterium (Fig. 3 B:4) immune mouse spleen cell can produce higher IFN-γ, compare restructuring hemolysin secretion bacterium (Fig. 3 B:3), the IFN-γ that restructuring III type secretion bacterium (Fig. 3 B:4) immune mouse produces is higher.
The present invention has also further evaluated the protection of oral each recombinant bacterium to schistosomicide: oral immunization in mice PBS, unloaded bacterium and recombiant vaccine bacterium three times; interval biweekly; after last immunity, one week subcutaneous abdomen infects 40 of schistosoma japonicum cercariaes; infect and within latter 42 days, calculate one-tenth borer population in Mice Body; worm reduction rate; liver worm's ovum number, egg reduction rate.Immunity PBS mouse become borer population be 30 ± 3 (Fig. 4 A:1) become borer population with unloaded bacterium be 26 ± 3 (Fig. 4 A:2), it is 20 ± 3 (Fig. 4 A:3) that restructuring hemolysin secretion bacterium becomes borer population, restructuring III type is secreted bacterium, and to become borer population be 17 ± 3 (Fig. 4 A:4); With respect to PBS immunity, the worm reduction rate of unloaded bacterium is 14.98%(Fig. 4 B:1), worm reduction rate 32.93%(Fig. 4 B:2 of restructuring hemolysin secretion bacterium), the worm reduction rate of restructuring III type secretion bacterium is 41.69%(Fig. 4 B:3); Immunity PBS mouse liver worm's ovum number is 122699 ± 34214(Fig. 4 C:1), unloaded bacterium liver worm's ovum number is 100398 ± 21669(Fig. 4 C:2), restructuring hemolysin secretion bacterium liver worm's ovum number is 73058 ± 25481(Fig. 4 C:3), restructuring III type secretion bacterium liver worm's ovum number is 51889 ± 12888(Fig. 4 C:4); With respect to PBS immunity, the egg reduction rate of unloaded bacterium is 18.17%(Fig. 4 D:1), restructuring hemolysin secretion bacterium egg reduction rate is 40.46%(Fig. 4 D:2), restructuring III type secretion bacterium egg reduction rate is 57.71%(Fig. 4 D:3).
In sum, the invention provides a kind of Schistosoma japonicum oral vaccine and application thereof that utilizes attenuation salmonella excretory system to express, this blood fluke vaccine oral vaccine, taking attenuation salmonella as carrier, is broken through the restriction of imitated vesicle structure for antigen presentation by the mode of secreting, expressing vaccine antigen.The attenuation salmonella oral vaccine submission system of secreting, expressing of the present invention can effectively be expressed and submission antigen in antigen presenting cell, and has good body internal stability.This vaccine submission system can activate body and produce the immune response for antigen molecule, obtains good protectiveness efficiency.
Above-described specific embodiment; object of the present invention, technical scheme and beneficial effect are further described; institute is understood that; the foregoing is only specific embodiments of the invention; be not limited to the present invention; within the spirit and principles in the present invention all, any amendment of making, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
<110> OrganizationName: Nanjing University
<120> Title: a kind of Schistosoma japonicum oral vaccine and application of attenuation salmonella secreting, expressing
<160> 14
<210> 1
<211> Length : 312
SequenceName : 1
SequenceDescription: III type secretion signal sopE
<212> Type : DNA
<213> OrganismName : Salmonella typhimurium
<400> PreSequenceString :
atgactaaca taacactatc cacccagcac tacagaatcc atagaagtga cgttgaacca 60
gtaaaagaaa aaacaacgga gaaggacatt tttgcaaaaa gtattactgc cgttagaaat 120
agctttatca gcctgtcgac gagtctgtca gatcgtttta gcctgcatca acaaacagac 180
ataccgacta cccattttca tcgtgggaac gcttctgagg gtagggcggt attaaccagt 240
aaaactgtta aagattttat gctgcaaaag ctcaatagtc tggatatcaa aggtaatgcg 300
agtaaagatc cg 312
<210> 2
<211> Length : 102
SequenceName : 2
SequenceDescription: I type secretion signal hlyN
<212> Type : DNA
<213> OrganismName : Escherichia coli
<400> PreSequenceString :
atgacaacaa taaccactgc acaaattaaa agcacactgc agtctgcaaa gcaatccgct 60
gcaaataaat tgcactcagc aggacaaagc acgaaagatg ca 102
<210> 3
<211> Length : 183
SequenceName : 3
SequenceDescription: I type secretion signal hlyC
<212> Type : DNA
<213> OrganismName : Escherichia coli
<400> PreSequenceString :
ttagcctatg gaagtcaggg tgatcttaat ccattaatta atgaaatcag caaaatcatt 60
tcagctgcag gtagcttcga tgttaaagag gaaagaactg cagcttcttt attgcagttg 120
tccggtaatg ccagtgattt ttcatatgga cggaactcaa taaccctgac cacatcagca 180
taa 183
<210> 4
<211> Length : 382
SequenceName : 4
SequenceDescription: nirB promotor
<212> Type : DNA
<213> OrganismName : Escherichia coli
<400> PreSequenceString :
ggttaccggc ccgatcgttg aacatagcgg tccgcaggcg gcactgctta cagcaaacgg 60
tctgtacgct gtcgtctttg tgatgtgctt cctgttaggt ttcgtcagcc gtcaccgtca 120
gcataacacc ctgacctctc attaattgct catgccggac ggcactatcg tcgtccggcc 180
ttttcctctc ttcccccgct acgtgcatct atttctataa acccgctcat tttgtctatt 240
ttttgcacaa acatgaaata tcagacaatt ccgtgactta agaaaattta tacaaatcag 300
caatataccc attaaggagt atataaaggt gaatttgatt tacatcaata agcggggttg 360
ctgaatcgtt aaggtaggcg gt 382
<210> 5
<211> Length : 891
SequenceName : 5
SequenceDescription : sj23LHD-GST
<212> Type : DNA
<213> OrganismName : Schistosoma japonicum
<400> PreSequenceString :
tacaaggata aaatcgatga cgaaattaat acactaatga ctggtgctct ggaaaatcca 60
aacgaggaaa taacggcaac catggataag atacaaacat cattccattg ttgtggagtc 120
aaaggtccag acgattataa agggaatgtg ccagcatcat gtaaagaagg gcaagaagtt 180
tatgttcagg gttgtctatc tgtctttagt gcattcttaa aacgcaactt aatgtcccct 240
atactaggtt attggaaaat taagggcctt gtgcaaccca ctcgacttct tttggaatat 300
cttgaagaaa aatatgaaga atggcatttg tatgagcgcg atgaaggtga taaatggcga 360
aacaaaaagt ttgaattggg tttggagttt cccaatcttc cttattatat tgatggtgat 420
gttaaattaa cacagtctat ggccatcata cgttatatag ctgacaagca caacatgttg 480
ggtggttgtc caaaagagcg tgcagagatt tcaatgcttg aaggagcggt tttggatatt 540
agatacggtg tttcgagaat tgcatatagt aaagactttg aaactctcaa agttgatttt 600
cttagcaagc tacctgaaat gctgaaaatg ttcgaagatc gtttatgtca taaaacatat 660
ttaaatggtg atcatgtaac ccatcctgac ttcatgttgt atgacgctct tgatgttgtt 720
ttatacatgg acccaatgtg cctggatgcg ttcccaaaat tagtttgttt taaaaaacgt 780
attgaagcta tcccacaaat tgataagtac ttgaaatcca gcaagtatat agcatggcct 840
ttgcagggct ggcaagccac gtttggtggt ggcgaccatc ctccaaaata a 891
<210> 6
<211> Length : 29
SequenceName : 6
SequenceDescription: III type secretion signal sopE P1
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
cccggatcca tgactaacat aacactatc 29
<210> 7
<211> Length : 26
SequenceName : 7
SequenceDescription: III type secretion signal sopE P2
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
aaaggtaccc ggatctttac tcgcat 26
<210> 8
<211> Length : 26
SequenceName : 8
SequenceDescription : nirB P1
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
cccctcgagg gttaccggcc cgatcg 26
<210> 9
<211> Length : 29
SequenceName : 9
SequenceDescription : nirB P2
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
cccggatcca ccgcctacct taacgattc 29
<210> 10
<211> Length : 31
SequenceName : 10
SequenceDescription: III type secretion sj23LHD-GST P1
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
aaaggtacca tgtacaagga taaaatcgat g 31
<210> 11
<211> Length : 36
SequenceName : 11
SequenceDescription: III type secretion sj23LHD-GST P2
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
taagttgcgt tttaagaatg ctagtatagg ggacat 36
<210> 12
<211> Length : 36
SequenceName : 12
SequenceDescription: III type secretion sj23LHD-GST P3
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
ttcttaaaac gcaacttaat gtcccctata ctaggt 36
<210> 13
<211> Length : 29
SequenceName : 13
SequenceDescription: III type secretion sj23LHD-GST P4
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
cccaagcttt tattttggag gatggtcgc 29
<210> 14
<211> Length : 27
SequenceName : 14
SequenceDescription: I type secretion si23LHD-GST P1
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
ccaatgcatc gtacaaggat aaaatcg 27
<210> 15
<211> Length : 27
SequenceName : 15
SequenceDescription: I type secretion si23LHD-GST P2
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
ccaatgcatc ttttggagga tggtcgc 27
<110> OrganizationName: Nanjing University
<120> Title: a kind of Schistosoma japonicum oral vaccine and application of attenuation salmonella secreting, expressing
<160> 14
<210> 1
<211> Length : 312
SequenceName : 1
SequenceDescription: III type secretion signal sopE
<212> Type : DNA
<213> OrganismName : Salmonella typhimurium
<400> PreSequenceString :
atgactaaca taacactatc cacccagcac tacagaatcc atagaagtga cgttgaacca 60
gtaaaagaaa aaacaacgga gaaggacatt tttgcaaaaa gtattactgc cgttagaaat 120
agctttatca gcctgtcgac gagtctgtca gatcgtttta gcctgcatca acaaacagac 180
ataccgacta cccattttca tcgtgggaac gcttctgagg gtagggcggt attaaccagt 240
aaaactgtta aagattttat gctgcaaaag ctcaatagtc tggatatcaa aggtaatgcg 300
agtaaagatc cg 312
<210> 2
<211> Length : 102
SequenceName : 2
SequenceDescription: I type secretion signal hlyN
<212> Type : DNA
<213> OrganismName : Escherichia coli
<400> PreSequenceString :
atgacaacaa taaccactgc acaaattaaa agcacactgc agtctgcaaa gcaatccgct 60
gcaaataaat tgcactcagc aggacaaagc acgaaagatg ca 102
<210> 3
<211> Length : 183
SequenceName : 3
SequenceDescription: I type secretion signal hlyC
<212> Type : DNA
<213> OrganismName : Escherichia coli
<400> PreSequenceString :
ttagcctatg gaagtcaggg tgatcttaat ccattaatta atgaaatcag caaaatcatt 60
tcagctgcag gtagcttcga tgttaaagag gaaagaactg cagcttcttt attgcagttg 120
tccggtaatg ccagtgattt ttcatatgga cggaactcaa taaccctgac cacatcagca 180
taa 183
<210> 4
<211> Length : 382
SequenceName : 4
SequenceDescription: nirB promotor
<212> Type : DNA
<213> OrganismName : Escherichia coli
<400> PreSequenceString :
ggttaccggc ccgatcgttg aacatagcgg tccgcaggcg gcactgctta cagcaaacgg 60
tctgtacgct gtcgtctttg tgatgtgctt cctgttaggt ttcgtcagcc gtcaccgtca 120
gcataacacc ctgacctctc attaattgct catgccggac ggcactatcg tcgtccggcc 180
ttttcctctc ttcccccgct acgtgcatct atttctataa acccgctcat tttgtctatt 240
ttttgcacaa acatgaaata tcagacaatt ccgtgactta agaaaattta tacaaatcag 300
caatataccc attaaggagt atataaaggt gaatttgatt tacatcaata agcggggttg 360
ctgaatcgtt aaggtaggcg gt 382
<210> 5
<211> Length : 891
SequenceName : 5
SequenceDescription : sj23LHD-GST
<212> Type : DNA
<213> OrganismName : Schistosoma japonicum
<400> PreSequenceString :
tacaaggata aaatcgatga cgaaattaat acactaatga ctggtgctct ggaaaatcca 60
aacgaggaaa taacggcaac catggataag atacaaacat cattccattg ttgtggagtc 120
aaaggtccag acgattataa agggaatgtg ccagcatcat gtaaagaagg gcaagaagtt 180
tatgttcagg gttgtctatc tgtctttagt gcattcttaa aacgcaactt aatgtcccct 240
atactaggtt attggaaaat taagggcctt gtgcaaccca ctcgacttct tttggaatat 300
cttgaagaaa aatatgaaga atggcatttg tatgagcgcg atgaaggtga taaatggcga 360
aacaaaaagt ttgaattggg tttggagttt cccaatcttc cttattatat tgatggtgat 420
gttaaattaa cacagtctat ggccatcata cgttatatag ctgacaagca caacatgttg 480
ggtggttgtc caaaagagcg tgcagagatt tcaatgcttg aaggagcggt tttggatatt 540
agatacggtg tttcgagaat tgcatatagt aaagactttg aaactctcaa agttgatttt 600
cttagcaagc tacctgaaat gctgaaaatg ttcgaagatc gtttatgtca taaaacatat 660
ttaaatggtg atcatgtaac ccatcctgac ttcatgttgt atgacgctct tgatgttgtt 720
ttatacatgg acccaatgtg cctggatgcg ttcccaaaat tagtttgttt taaaaaacgt 780
attgaagcta tcccacaaat tgataagtac ttgaaatcca gcaagtatat agcatggcct 840
ttgcagggct ggcaagccac gtttggtggt ggcgaccatc ctccaaaata a 891
<210> 6
<211> Length : 29
SequenceName : 6
SequenceDescription: III type secretion signal sopE P1
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
cccggatcca tgactaacat aacactatc 29
<210> 7
<211> Length : 26
SequenceName : 7
SequenceDescription: III type secretion signal sopE P2
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
aaaggtaccc ggatctttac tcgcat 26
<210> 8
<211> Length : 26
SequenceName : 8
SequenceDescription : nirB P1
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
cccctcgagg gttaccggcc cgatcg 26
<210> 9
<211> Length : 29
SequenceName : 9
SequenceDescription : nirB P2
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
cccggatcca ccgcctacct taacgattc 29
<210> 10
<211> Length : 31
SequenceName : 10
SequenceDescription: III type secretion sj23LHD-GST P1
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
aaaggtacca tgtacaagga taaaatcgat g 31
<210> 11
<211> Length : 36
SequenceName : 11
SequenceDescription: III type secretion sj23LHD-GST P2
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
taagttgcgt tttaagaatg ctagtatagg ggacat 36
<210> 12
<211> Length : 36
SequenceName : 12
SequenceDescription: III type secretion sj23LHD-GST P3
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
ttcttaaaac gcaacttaat gtcccctata ctaggt 36
<210> 13
<211> Length : 29
SequenceName : 13
SequenceDescription: III type secretion sj23LHD-GST P4
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
cccaagcttt tattttggag gatggtcgc 29
<210> 14
<211> Length : 27
SequenceName : 14
SequenceDescription: I type secretion si23LHD-GST P1
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
ccaatgcatc gtacaaggat aaaatcg 27
<210> 15
<211> Length : 27
SequenceName : 15
SequenceDescription: I type secretion si23LHD-GST P2
<212> Type : DNA
<213> OrganismName : Artificial
<400> PreSequenceString :
ccaatgcatc ttttggagga tggtcgc 27
Claims (2)
1. a blood fluke vaccine Salmonellas secretion expression carrier, it is characterized in that by the control of bacterium promotor, Salmonellas secretion effect protein and secretion signal guiding thereof, the recombinant salmonella expression vector of secreting, expressing schistosome antigen, described bacterium promotor is Salmonellas hemolysin promotor, described Salmonellas secretion effect protein refers to hlyA, hlyB, hlyC, hlyD, described secretion signal refers to 34 amino acid of Salmonellas hlyA albumen n end and 61 aminoacid sequences of C end, described schistosome antigen refers to Schistosoma japonicum bivalent antigen sj23LHDGST, sj23LHDGST is the large hydrophilic area of sj23 molecule and the fusion rotein of GST.
2. the application of blood fluke vaccine Salmonellas secretion expression carrier claimed in claim 1 in preparation Schistosoma japonicum oral vaccine.
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| CN201110232776.2A CN102277373B (en) | 2011-08-15 | 2011-08-15 | Schistosoma japonica vaccine expression vector for attenuated salmonella secretion expression and use thereof |
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| CN103204936B (en) * | 2013-01-25 | 2014-06-18 | 天津商业大学 | Preparation method for polyclonal antibody to salmonella effect protein SopB |
| CN104805113A (en) * | 2015-05-06 | 2015-07-29 | 南昌大学 | Application of attenuated salmonella typhimurium VNP20009 in gene expression |
| CN114480462A (en) * | 2020-10-26 | 2022-05-13 | 南京吉芮康生物科技研究院有限公司 | Novel coronavirus vaccine antigen presentation system for secretion expression of NTD structural domain protein by attenuated salmonella and application thereof |
| CN114703214A (en) * | 2022-03-03 | 2022-07-05 | 南京吉芮康生物科技研究院有限公司 | Novel loss-resistant spatiotemporal controllable expression plasmid and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996014871A1 (en) * | 1994-11-15 | 1996-05-23 | Cortecs Limited | Immunogenic compositions |
| CN102335421A (en) * | 2011-08-03 | 2012-02-01 | 南京大学 | Attenuated salmonella inducible secretory expression oral vaccine presentation system and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996014871A1 (en) * | 1994-11-15 | 1996-05-23 | Cortecs Limited | Immunogenic compositions |
| CN102335421A (en) * | 2011-08-03 | 2012-02-01 | 南京大学 | Attenuated salmonella inducible secretory expression oral vaccine presentation system and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Guo Chen等.Oral Delivery of the Sj23LHD-GST Antigen by Salmonella typhimurium Type III Secretion System Protects against Schistosoma japonicum Infection in Mice.《plos negl trop dis》.2011,第5卷(第9期),1-11. * |
| 曾凡杰等.噬菌体随机肽库筛选日本血吸虫22.6kDa蛋白有效抗原表位的初步研究.《东南大学学报(医学版)》.2003,第1卷(第22期),6-8,25. * |
| 李浩等.肿瘤靶向性沙门氏菌联合环磷酰胺治疗裸鼠Tca8113移植瘤的实验研究.《口腔医学研究》.2010,第26卷(第4期),493-496. * |
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