CN109942693A - A kind of CTL epitope polypeptide of Cryptosporidum parvum and its application and vaccine - Google Patents
A kind of CTL epitope polypeptide of Cryptosporidum parvum and its application and vaccine Download PDFInfo
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- CN109942693A CN109942693A CN201910266528.6A CN201910266528A CN109942693A CN 109942693 A CN109942693 A CN 109942693A CN 201910266528 A CN201910266528 A CN 201910266528A CN 109942693 A CN109942693 A CN 109942693A
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- polypeptide
- cryptosporidum parvum
- ctl epitope
- nucleic acid
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to a kind of CTL epitope polypeptides of Cryptosporidum parvum, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO.1.The nucleic acid of the CTL epitope polypeptide of the code Cryptosporidum parvum is also provided, the nucleotide sequence of the nucleic acid is as shown in SEQ ID NO.2.The CTL epitope polypeptide P1 of Cryptosporidum parvum of the present invention, has good affinity with H-2Kb molecule or HLA-A*0201 molecule respectively, illustrates there is corresponding immunologic function.A kind of new direction is provided to prepare Cryptosporidum parvum vaccine, and the polypeptide shows excellent irritative response in polypeptid specificity CTL immune response in C57BL/6N mouse boosting cell, has huge development and application potentiality in Cryptosporidum parvum specific active immunotherapy field.
Description
Technical field
The invention belongs to biomolecule technical field, it is related to CTL epitope polypeptide and its application of a kind of Cryptosporidum parvum
And vaccine.
Background technique
Cryptosporidum parvum (Cryptosporidium parvum), which is under the jurisdiction of, pushes up multiple door (PhylumApicomplexa)
Sporozoa (ClassSporozoa) was reported and was named by Tyzzer for the first time in 1912, was a kind of important infecting both domestic animals and human
Protozoon.In reported 31 Zoonosis Cryptosporidium type, can infect people and most of mammals is small hidden
Sporozoite, and Cryptosporidum parvum host is the most extensive, mainly include people, Bovidae, various rodent mammals, Camelidae,
Equine, Canidae, non-human primates and marine mammal and fish.According to incompletely statistics, between 2004-2010, the world is multiple
Country fulminant and sporadic Cryptosporidium case because of caused by water biography or food transmission have reached hundreds of.Cryptosporidum parvum
Can long-term surviving in the environment, and general sewage water treatment method such as filters and hypochlorous acid disinfection cannot effectively kill water
In egg capsule, stability of the egg capsule in water environment and low infective dose (being less than 10 egg capsules) are that water source Cryptosporidium is infected
Two major reasons.Cryptosporidum parvum infection is one of the important pathogen for leading to humans and animals diarrhoeal diseases, annual big
There are about 8,000,000 five years old or less children to die of the diarrhea as caused by Cryptosporidium spp.Clinical and epidemiological survey the results show that
Cryptosporidum parvum has become common one of the secondary infection parasitic protozoa of aids patient, long-term severe diarrhea, and leads
Cause one of the key factor of malnutritive and immunologic inadequacy AIDS patients death.In addition, Cryptosporidum parvum is in
It infects also very universal in poultry, if infection rate is up to 19.5% to Cryptosporidum parvum in calf before wean, causes livestock and poultry to increase slow
Slowly, immunity decrease, other secondary pathogen infections, to endanger young age livestock and poultry life.Therefore, the prevention and control of Cryptosporidum parvum have been
As the outstanding problem in the fields such as animal husbandry, public health, the specific medicament and vaccine of prevention and treatment Cryptosporidiosis there is no at present.
The specific cellular immunity that cytotoxic T lymphocyte (Cytotoxic T lymphocytes, CTL) mediates is answered
It answers and plays key effect during body resists microorganism infection intracellular.After Cryptosporidium parvum Oocysts infection cell, table
The albumen such as face GP15/40, CP15, CP23 pass through a series of enzymolysis process infected into the cell, are degraded to 8-11 amino acid
Polypeptide, then with corresponding major histocompatibility complex (Major histocompatibility complex, MHC) I
Class molecular antigen polypeptide engagement groove combines, and forms MHC I- antigenic peptide complexes, and be presented to infection cell surface.MHC I- is anti-
Former peptide complex (pMHCI) be expressed in T cell surface receptor (T cell receptor, TCR) identification and it is in combination.
Cytotoxic T lymphocyte (CTL) can dissolve infection cell in gastrointestinal tract by release perforin or granzyme, discharge prematurity
Cyst, cutting Cryptosporidium breeding in the cell, to effectively control the infection of Cryptosporidium.Studies have shown that and body
Liquid immune response is compared, and cellullar immunologic response has prior effect, and CD in anti-Cryptosporidium spp4+T cell and CD8 +T cell all has important function in anti-Cryptosporidum parvum (C.parvum) is immune.Such as Pantenburg is to people CD8+T
It is found in the functional study that Cryptosporidum parvum infects in cell clearance enterocyte, I molecule of people HLA can be by presenting hidden spore
The immunodominant epitope of sub- GP15 albumen activates CD8+T cellullar immunologic response.
During the infection immunity of Cryptosporidium, surface protein GP15/40, CP15 and CP23 albumen all have induction
The ability of cellullar immunologic response, wherein CP15, CP23 albumen are in Cryptosporidum parvum (C.parvum) and people Cryptosporidium
(C.hominis) homology is up to 95% or more between genotype, and sequence is relatively conservative, can be used as the conservative T cell table of screening
The target protein of position.In addition, CircumSporozoite sample glycoprotein C SL (circumsporozoite-like glycoprotein), heat are stopped
Gram albumen HSP70 and surface glycoprotein GP900 adheres to and invades the important albumen of host body as Cryptosporidium, and also have can for pole
The potential target site for preventing and treating Cryptosporidiosis can be become.Pass through the identification to Cryptosporidum parvum CTL epitope, screening
Relatively conservative CTL epitope out, provides significant data for the research of polypeptide vaccine.Currently, about Cryptosporidum parvum CTL epitope
Most study is people's HLA-B and HLA-C genotype, and the HLA-A* of other genotype wide expression such as in crowd to people
The research of 0201 molecule and the MHC molecule of animal is less, hinders the research about Cryptosporidum parvum epiposition vaccine and answers
With.
Summary of the invention
In view of this, one of the objects of the present invention is to provide a kind of CTL epitope polypeptides of Cryptosporidum parvum.
In order to achieve the above objectives, the invention provides the following technical scheme:
1. a kind of CTL epitope polypeptide of Cryptosporidum parvum, the amino acid sequence of the polypeptide is as shown in SEQ ID NO.1.
2. the present invention also provides a kind of CTL tables of encoding amino acid sequence Cryptosporidum parvum as shown in SEQ ID NO.1
The nucleic acid of position polypeptide.
Further, the nucleotide sequence of the nucleic acid is as shown in SEQ ID NO.2.
3. the third object of the present invention be to provide the recombinant expression carrier containing nucleic acid, expression cassette, transgenic cell line,
Recombinant bacterium or recombinant viral vector, nucleic acid energy encoding amino acid sequence Cryptosporidum parvum as shown in SEQ ID NO.1
CTL epitope polypeptide.
Further, the nucleotide sequence of the nucleic acid is as shown in SEQ ID NO.2.
Nucleic acid of the invention can be rna form (such as mRNA, hnRNA, tRNA or any other form), be also possible to
DNA form (including but not limited to by clone or be synthetically produced, or any combination thereof and generate cDNA and genomic DNA).
DNA can be three chains, double-strand or single-stranded, or any combination thereof.The arbitrary portion of at least one chain of DNA or RNA can be volume
Code chain, also referred to as sense strand, or can be noncoding strand, also referred to as antisense strand.
4. nucleic acid described in the CTL epitope polypeptide of Cryptosporidum parvum described in technical solution 1, technical solution 2, technical solution 3
The recombinant expression carrier, expression cassette, transgenic cell line, recombinant bacterium or recombinant viral vector are preparing Cryptosporidum parvum epidemic disease
Application in seedling.
5. a kind of Cryptosporidum parvum vaccine, the active constituent of the Cryptosporidum parvum vaccine be in following substance extremely
Few one kind:
A. the CTL epitope polypeptide of Cryptosporidum parvum, amino acid sequence is as shown in SEQ ID NO.1；
B. the nucleic acid of the CTL epitope polypeptide of encoding amino acid sequence Cryptosporidum parvum as shown in SEQ ID NO.1；
C. nucleotide sequence nucleic acid as shown in SEQ ID NO.2；
D. recombinant expression carrier, expression cassette described in technical solution 3, transgenic cell line, recombinant bacterium or recombinant virus carry
Body.
Further, the vaccine further includes one or more vaccine adjuvants.
Cryptosporidum parvum vaccine of the invention can pass through injection, injection, collunarium, eye drip, infiltration, absorption, physics or change
It learns the method mediated and imports body such as muscle, intradermal, subcutaneous, vein, mucosal tissue；Or by after other material mixings or package
Import body.
6. the present invention also provides cores described in the CTL epitope polypeptide of Cryptosporidum parvum described in technical solution 1, technical solution 2
It is prepared by recombinant expression carrier, expression cassette, transgenic cell line, recombinant bacterium or recombinant viral vector described in acid, technical solution 3
CD8+Application in T lymphocyte multiplication agent.
The beneficial effects of the present invention are: a kind of CTL epitope polypeptide P1 of Cryptosporidum parvum, the polypeptide and H- are provided
2Kb molecule or HLA-A*0201 molecule have good affinity respectively, illustrate there is corresponding immunologic function.It is small to prepare
Cryptosporidium vaccine provides a kind of new direction, and polypeptide polypeptid specificity CTL in C57BL/6N mouse boosting cell is immune anti-
Excellent irritative response, induced CD are shown in answering8+The IFN-gamma that T cell is generated is in various negative control ratios
Compared with P < 0.001 has extremely significant sex differernce, illustrates the CTL epitope polypeptide P1 of Cryptosporidum parvum as vaccine activity substance
Energy effective stimulus body reaction, and specific secretion goes out IFN-gamma, further has lethal effect to Cryptosporidum parvum,
There are huge development and application potentiality in Cryptosporidum parvum specific active immunotherapy field.
Detailed description of the invention
In order to keep the purpose of the present invention, technical scheme and beneficial effects clearer, the present invention provides following attached drawing and carries out
Illustrate:
Fig. 1 is the SDS-PAGE qualification figure of H-2Kb protein mini-expression inducing expression；
Fig. 2 is the SDS-PAGE qualification figure of m β 2m protein mini-expression inducing expression；
Fig. 3 is sieve chromatography result and electrophoresis after H-2Kb, m β 2m and polypeptide P1 renaturation；
Fig. 4 is sieve chromatography result and electrophoresis after HLA-A*0201, m β 2m and polypeptide P1 renaturation；
Fig. 5 is respectively to detect knot with the ELISPOT of 8 polypeptid specificity CTL immune responses in C57BL/6N mouse boosting cell
Fruit；
Fig. 6 is the ELISPOT testing result of P1 polypeptid specificity CTL immune response in C57BL/6N mouse boosting cell, separately
Four control groups of outer setting: unrelated polypeptide group, there are also adjuvant group, DMSO group, negative control groups.
Specific embodiment
The principle of the present invention, feature and preferred embodiment are described in detail below in conjunction with attached drawing, example is only
It is used to explain the present invention, is not intended to limit the scope of the present invention.Test method without specific conditions in embodiment, usually
According to normal conditions or according to the normal condition proposed by manufacturer.Test material as used in the following examples, such as without special theory
It is bright, it is to be commercially available from routine biochemistry reagent shop.Quantitative test in following embodiment, is respectively provided with and repeats reality three times
It tests, results are averaged.
Embodiment 1
The selection and synthesis of Cryptosporidum parvum Gp15 protein CTL epitope polypeptide.
To the principal causative albumen and surface protein GP15, GP40, CSL, GP900, HSP70, CP15 of Cryptosporidum parvum
It is scanned with CP23, chooses the polypeptide of amino acid sequence such as SEQ ID No.1, be named as P1, nucleotide sequence such as sequence
In table shown in SEQ ID No.2, studied.Polypeptide P1 derives from GP15 albumen, using solid phase polypeptide synthesis (Fmoc/tBu
Strategy) synthesis polypeptide P1, purified after synthesis with HPLC, purity 98%.
The present invention applies bioinformatic analysis method to predict the restricted polypeptide of H-2Kb, H-2Db first, and from these
A large amount of polypeptide has been screened in epitope to be tested, and 8 stronger 8 polypeptide (peptide1- of prediction affinity are finally filtered out
Peptide8, peptide2 are the polypeptide of amino acid sequence such as SEQ ID No.1, are named as P1), purified after synthesis with HPLC.
Embodiment 2
With the refolding experiment detection Cryptosporidum parvum CTL epitope polypeptide and mouse H-2kb and people HLA-A* of albumen
The binding ability of 0201 molecule
One, the preparation of mouse MHC I class molecule (H-2Kb) and light chain β 2m (mouse β 2m, m β 2m) inclusion body protein
By encoding murine MHC I class molecule heavy chain (H-2kb) (C57BL/6N, GenBank No.XM_017317259.1)
DNA molecular (the nucleotide sequence such as sequence table of extracellular region amino acid (amino acid sequence is as shown in SEQ ID No.3 in sequence table)
Shown in middle SEQ ID No.4) it is inserted between prokaryotic expression plasmid pET-21a (+) restriction enzyme site Nde I and Xho I, obtain weight
Group expression plasmid, is named as pET-21a (+)/H-2kb.
The pET-21a (+) of building/H-2Kb recombinant expression plasmid is converted into e. coli bl21 (ED3) competent cell,
37 DEG C of overnight incubations utilize ampicillin (Amp) resistance screening recombinant conversion body.Positive restructuring bacterium is picked them separately (to be named as
BL21 (ED3)/H-2Kb) single colonie, be inoculated in LB culture medium of the 100mL containing Amp.After 37 DEG C of shake cultures,
According to 1:100 dilution be inoculated in the triangular flask containing 2L fresh LB, 37 DEG C, 200rpm shaking culture extremely
Logarithmic growth phase (OD600=0.4~0.6).1mL bacteria suspension is taken out, 4000rpm is centrifuged 3min, discards supernatant, with 50 μ L loadings
Buffer be resuspended bacterial precipitation, -20 DEG C freeze it is spare.Then IPTG to final concentration of 1mmol/L, induction are added in the medium
1mL is taken out after cultivating 4 hours, 4000rpm is centrifuged 3min and collects thallus, 5 × SDS sample-loading buffer is added after abandoning supernatant, with
The culture sample (control) acquired before induction boils 5min simultaneously, expresses feelings with 12% polyacrylamide gel electrophoresis inspection
Condition.As a result, it was confirmed that expression bacterium BL21 (ED3)/H-2Kb containing recombinant expression plasmid can express SEQ ID No.3 institute after inducing
The albumen shown, is named as H-2Kb.H-2Kb protein mini-expression inducing expression SDS-PAGE electrophoresis is as shown in Figure 1.
Two, by BL21 (ED3)/H-2Kb bacterium solution of the polyacrylamide gel electrophoresis detection through above-mentioned 12%, continue to train
It supports, cultured bacterium solution is moved into the centrifuge tube of 300mL, 4 DEG C of 5000rpm centrifugation 10min abandon supernatant.Thallus is collected to add in right amount
PBS buffer solution is resuspended thallus, sonicated cells, ultrasonic 6sec, is spaced 10sec, and totally 99 times, between power 200~400.It is super
Sound, which has cracked, is transferred to 50mL centrifuge tube, and 11000rpm is centrifuged 30min, scratches the cell fragment on upper layer (in precipitating with glass rod with gentle
Lower layer density is all cell fragment), then outwell supernatant.Washing buffer be resuspended, measure it is indefinite, view inclusion body how much and
Fixed, general 35mL, ultrasonic 4sec are spaced 10s, 30 times, repeat the above steps 2 times.Buffer is resuspended, mixing, 11000rpm is resuspended
It is centrifuged 30min, blows away the fragment in precipitating.Resulting mouse MHC I class molecule heavy chain H-2Kb inclusion body protein, is dissolved in salt
In sour guanidine solution, concentration 30mg/ml.
It is obtained using method identical with mouse MHC I class molecule H-2kb inclusion body protein is obtained comprising mouse MHC I class
Molecule light chain β 2m (mouse β 2m, m β 2m), by the 1-98 amino acids of its mature peptide gene (in amino acid sequence such as sequence table
Shown in SEQ ID No.5) DNA (nucleotide sequence is as shown in SEQ ID No.6 in sequence table) be inserted into prokaryotic expression plasmid
Between pET-21a (+) restriction enzyme site Nde I and Xho I, recombinant expression plasmid is obtained, is named as pET-21a (+)/m β 2m.Contain
Expression bacterium BL21 (ED3)/pET-21a (+) of recombinant expression plasmid/m β 2m can be expressed after inducing shown in SEQ ID No.5
Albumen is named as m β 2m.It finally obtains inclusion body protein to be dissolved in guanidine hydrochloride solution, is named as m β 2m, wrap
Culvert bulk concentration is 30mg/ml.M β 2m protein mini-expression inducing expression SDS-PAGE electrophoresis is as shown in Figure 2.
Three, the refolding of H-2kb, m β 2m and peptide
By the inclusion body protein and polypeptide P1 of mouse MHC I class molecule heavy chain (H-2Kb) and light chain (m β 2m) according to rubbing
You are added in renaturing inclusion bodies reagent (Refolding Buffer) ratio than 1:1:3.Details are provided below:
(1) that renaturing inclusion bodies reagent (general 500ml system, specifically depending on renaturation effect) is placed in 4 DEG C of refrigerators is (or cold
In library) pre-cooling.Renaturation process carries out (4 DEG C of refrigerators or freezer) under the conditions of 4 DEG C.(2) renaturing inclusion bodies reagent will be filled
Beaker is placed on magnetic stirring apparatus, and magnetic rotor is added, suitable mixing speed is arranged (60rpm is advisable).(3) 1mL hydrochloric acid is taken
The dissolved m β 2m inclusion body (30mg/ml) of guanidine is added in syringe, is allowed to be slowly dropped into renaturing inclusion bodies reagent, slowly stir
Mix 6-8h.(4) 5mg polypeptide P1 is dissolved in 200 μ L DMSO, is then added directly into renaturing inclusion bodies reagent with pipettor
In.(5) after slowly stirring 30min, in 3mL H-2kb inclusion body (30mg/ml) is added in syringe, 8-12h is slowly stirred.It can
Action time is appropriately extended.
Four, the concentration of complex proteins
After renaturation, carefully renaturation solution is moved in 4 DEG C of refrigerators or freezer in pressure stirring-type concentration cup, using nitrogen
The molecular sieve buffering of 120mL pre-cooling is added when the concentrate solution on 10kDa ultrafiltration membrane to volume is about 30mL for pressure for air lift
Liquid.Solution is transferred in 50mL centrifuge tube when being finally concentrated into 30mL or so, 4 DEG C, 12000rpm, which is centrifuged 15min and removes, to sink
It forms sediment, supernatant is transferred in 10KD protein concentration pipe, 4 DEG C, 2400rpm is further concentrated into 2-5ml.If renaturation solution after renaturation
Become muddy, can be after low-temperature centrifugation removal precipitating, then be concentrated with pressure stirring-type concentration cup.
Five, the sieve chromatography of complex proteins
200 16/60HiLoad gel chromatography column of Superdex is balanced with molecular sieve buffer.It is (general after balance is good
1ml/min needs 2h), by the complex proteins sample for being concentrated and changing after liquid finishes be centrifuged off precipitating (4 DEG C, 12000rpm,
10min), after the loading pump that fast protein liquid chromatography instrument (FPLC) is washed with molecular sieve buffer, supernatant is taken to inject.It is chromatographing
(general 1ml/min) is run with flow velocity appropriate on column, renaturation effect is detected according to the result of sieve chromatography, by corresponding molecule
The protein peak of amount is collected, and is identified through SDS-PAGE.
As a result as shown in figure 3, Fig. 3 be H-2Kb, m β 2m and polypeptide P1 (H-Kb molecular weight about 32KD, m β 2m 10KD or so,
P1 is a small peptide, be can be neglected) sieve chromatography result after renaturation.Wherein, A1 H-2Kb, m β 2m and polypeptide P1 are multiple
Sieve chromatography figure after property；A2 is the protein peak SDS-PAGE qualification result collected after sieve chromatography, label and peak label phase
It is corresponding；There are 2 main peaks altogether under the conditions of the UV absorption of 280nm in protein chromatographic, collects the albumen 15%SDS- at peak value
PAGE electrophoretic analysis, the peak 1 at 82mL elution volume is H-2kb/P1/m β 2m complex monomer, protein molecular as the result is shown
Amount is about 42kDa；Second main peak (peak 2) is m β 2m albumen in 105mL elution volume or so.
Using identical method by the inclusion body protein of people's MHC I class molecule heavy chain (HLA-A*0201) and light chain (m β 2m)
And polypeptide P1 according to molar ratio 1:1:3 ratio be added renaturing inclusion bodies reagent (Refolding Buffer) in, through renaturation,
Concentration, sieve chromatography identification, it was demonstrated that HLA-A*0201 also can be in conjunction with P1 polypeptide (result is shown in such as Fig. 4).Fig. 4 is HLA-A*
0201, sieve chromatography result after m β 2m and polypeptide P1 renaturation.Wherein, after A1 HLA-A*0201, m β 2m and polypeptide P1 renaturation
Sieve chromatography figure；A2 is corresponding with peak label for the protein peak SDS-PAGE qualification result label collected after sieve chromatography；
There are 2 main peaks altogether under the conditions of the UV absorption of 280nm in protein chromatographic, collects the albumen 15%SDS-PAGE electricity at peak value
Swimming analysis, the peak 1 at 82mL elution volume is HLA-A*020/P1/m β 2m complex monomer, molecular weight of albumen as the result is shown
About 42kDa；Second main peak (peak 2) is m β 2m albumen (about 10KDa) in 105mL elution volume or so.
It can be shown that H-2Kb molecule, HLA-A*0201 molecule have good parent with polypeptide P1 respectively by sieve chromatography result
And power, there is corresponding immunologic function.
Embodiment 3
Specific CTL immunity after mouse is immunized with ELISPOT method detection Cryptosporidum parvum CTL epitope polypeptide is answered
It answers.
MHC I/ antigenic peptide complexes are the key factors for activating specific CTL immunity response.C57BL/6N mouse MHC loses
Clear background is passed, can be used as the experimental animal model of research Cryptosporidum parvum cellullar immunologic response.Rasmussen etc. studies table
Bright, C57BL/6N mouse (its mhc gene type are as follows: H-2Kb, H-2Db) is in inoculation 106After a Cryptosporidium parvum Oocysts, whole
Infection Status is kept during a experiment, illustrates that the Strains of Mouse is related with the resistance of Cryptosporidium and neurological susceptibility.And H-2Kb is limited
Property epitope processed and the epitope of the HLA-A*0201 molecule of wide expression in crowd have similar anchor residues.Therefore, with
C57BL/6N mouse is that host screens avian influenza virus CTL epitope, then can be the research of anti-human Cryptosporidum parvum polypeptide vaccine
Reliable basis is provided.
One, experimental animal immune
Polypeptide P1 (being emulsified using Freund's complete adjuvant) the immune four week old C57BL/6N mouse prepared with embodiment 1.Altogether
It is 3 times immune, it carries out second within 7 days after first time is immune and is immunized, initial immunity uses Freund's complete adjuvant, second and third time
It is immune to use incomplete Freund's adjuvant.Each immunizing dose is 100ug polypeptide P1/, before immune group mouse initial immunity
Stomach-filling 2*106A Cryptosporidium parvum Oocysts.Latter all separating mouse spleen lymphocytes are immunized in third time, and carry out cytometer
Number.
Two, the separation of Mouse spleen cells
Cervical dislocation puts to death mouse, is put into 75% alcohol, spare；Separating spleen slowly removes spleen with tweezers；It will
Isolated spleen is added in RPMI-1640 culture medium, with syringe nozzle, is ground using the cell sieve of 40um；By lapping liquid
It slowly sucks in centrifuge tube, 1500rpm (350-400g)/min, 4 DEG C of centrifugation 5min abandon supernatant after centrifugation, flicked with index finger surplus
Extraction raffinate body 4-5 times, cell is hanged；Cell pyrolysis liquid 10ml is added, lysis at room temperature 10min, 1500rpm are centrifuged 5min；With
RPMI-1640 culture medium washs cell 1-2 times, removes red blood cell and connective tissue；Supernatant carefully moved to after centrifugation new
In 15ml centrifuge tube, 10ul cell count (the round white point of microscopically observation Cheng Liang) is taken.
Three, the separation of mouse CD4 and cd8 cell
Beads enrichment reagent is as follows:
MojoSort Mouse CD4T Cell Isolation Kit, producer: Biolegend article No.: 480006.
MojoSort Mouse CD8T Cell Isolation Kit, producer: Biolegend article No.: 480008.
Cell is resuspended with Molosort Buffer, 300g is centrifuged 5min；By 100ul cell suspension (107Cells) with
10ul Biotin-Antibody Cocktail is mixed, until being incubated for 15min on ice (in streaming pipe)；Cell is resuspended, 10ul is added
Streptavidin Nanobeads is mixed to 15min on ice；2.5ml mojosort Buffer is added；Streaming pipe is placed in
5min in magnet；Target cell is poured into new 15ml pipe, be centrifuged and carry out cell count.
Four, the response of mouse H-2b specific CTL immunity detects
ELISPOT screening is Mouse IFN-gamma ELISpotPLUS (HRP), producer: MabTech, article No.
3321-4HPT-2。
Take the lymphocyte prepared and the lymphocyte (about 10 of isolated CD4 and CD86Cells), it is plated on and handles well
ELISPOT plate in, be added polypeptide stimulant (10ug/ml), and set positive control (ConA) respectively, negative control (is not added
Peptide) and blank control (blank cultures)；It is placed in 37 DEG C of CO212-48h is cultivated in culture medium, it is immovable in the training period
ELISPOR plate；After culture, cell is discarded, is washed 5 times with PBS, 200ul is added in every hole；Biotinylated antibody is added
(1ug/ml), 37 DEG C of incubation 2h；PBS is washed 5 times, is added after HRP-Streptavidin in 37 DEG C of incubations 1h, PBS washing 5 times
Substrate colour developing is added afterwards；Room temperature is read after drying on ELISpot reading machine, as a result sees Fig. 5 and Fig. 6.
Fig. 5 is the result of the Cryptosporidium epitope of ELISPOT identification in vivo indirectly.Wherein, abscissa is 8 polypeptide (this hairs
The bright polypeptide P1 (polypeptide is not added with 7 polypeptides in addition tested together, peptide1-peptide8) and negative control
The cell of stimulation, control without peptide), Native mouse boosting cell compare (i.e. no small hidden spore of stomach-filling
The mouse boosting cell of worm's ovum capsule, Nagative control) and blank control (blank cultures, blank control), it indulges and sits
It is designated as spot formation cell quantity, with spot formation cell (spot forming cells) SFC/106It indicates.
The best polypeptide P1 polypeptide of immune response effect in Fig. 5 has been carried out mouse vivo immunization again by Fig. 6, is further led to
Cross the directly Cryptosporidium epitope of ELISPOT identification in vivo.After animal polypeptide is immune, separating Morr. cell uses MojoSort
Mouse CD8+T cell Isolation kit magnetic bead sorting CD8+T cell, 5 groups of experiment point carry out, including polypeptide P1 group,
(irrelevant peptide control, unrelated polypeptide is selected from H1N1 type influenza virus protein to unrelated polypeptide group
Epitope polypeptide), adjuvant group (adjuvant control), DMSO group (DMSO group control), negative control group
(Nagative control, mouse only stomach-filling Cryptosporidium parvum oocysts suspended, without immunologic process).Abscissa represents 5 groups, indulges and sits
It is designated as spot formation cell quantity, with spot formation cell (spot forming cells) SFC/106It indicates.
Can be seen that polypeptide P1 from Fig. 5, Fig. 6 can stimulate CD8+T cell generates very good immune response, each right
According to group almost without or a small amount of specific IFN-γ secretion, experimental group polypeptide P1 can cause very strong specific IFN-γ secretion,
Compared with various negative or other polypeptides, * * represents P < 0.001, and (P < 0.05 thinks there is statistical significance, P < for difference
0.001 indicates there is extremely significant difference), illustrate that there are 5 significant differences.Illustrate that polypeptide P1 can evoke stronger spy in vivo
Specific T cell immunity reaction can generate lethal effect to Cryptosporidum parvum.
The CTL epitope polypeptide P1 of Cryptosporidum parvum of the invention can be used as active constituent and pharmaceutically acceptable
Vehicle group is at composition, alternatively, with other extracts with pharmacological activity and/or synthetic drug and pharmaceutically acceptable load
Body forms composition, according still further to pharmaceutical field conventional method be made polypeptide P1 express positive Cryptosporidum parvum epidemic prevention or
Therapeutical peptide vaccine has huge development and application potentiality in Cryptosporidum parvum specific active immunotherapy field.
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Sequence table
<110>Zhoukou Normal University
<120>a kind of CTL epitope polypeptide of Cryptosporidum parvum and its application and vaccine
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Ser Val Phe Ala Ile Phe Ala Ala Ile Phe
1 5 10
<210> 2
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tcggtattcg cgagattcgc cgcaatcttt 30
<210> 3
<211> 275
<212> PRT
<213>mouse (Mus musculus)
<400> 3
Gly Pro His Ser Leu Arg Tyr Phe Val Thr Ala Val Ser Arg Pro Gly
1 5 10 15
Leu Gly Glu Pro Arg Tyr Met Glu Val Gly Tyr Val Asp Asp Thr Glu
20 25 30
Phe Val Arg Phe Asp Ser Asp Ala Glu Asn Pro Arg Tyr Glu Pro Arg
35 40 45
Ala Arg Trp Met Glu Gln Glu Gly Pro Glu Tyr Trp Glu Arg Glu Thr
50 55 60
Gln Lys Ala Lys Gly Asn Glu Gln Ser Phe Arg Val Asp Leu Arg Thr
65 70 75 80
Leu Leu Gly Tyr Tyr Asn Gln Ser Lys Gly Gly Ser His Thr Ile Gln
85 90 95
Val Ile Ser Gly Cys Glu Val Gly Ser Asp Gly Arg Leu Leu Arg Gly
100 105 110
Tyr Gln Gln Tyr Ala Tyr Asp Gly Cys Asp Tyr Ile Ala Leu Asn Glu
115 120 125
Asp Leu Lys Thr Trp Thr Ala Ala Asp Met Ala Ala Leu Ile Thr Lys
130 135 140
His Lys Trp Glu Gln Ala Gly Glu Ala Glu Arg Leu Arg Ala Tyr Leu
145 150 155 160
Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Lys Asn Gly Asn
165 170 175
Ala Thr Leu Leu Arg Thr Asp Ser Pro Lys Ala His Val Thr His His
180 185 190
Ser Arg Pro Glu Asp Lys Val Thr Leu Arg Cys Trp Ala Leu Gly Phe
195 200 205
Tyr Pro Ala Asp Ile Thr Leu Thr Trp Gln Leu Asn Gly Glu Glu Leu
210 215 220
Ile Gln Asp Met Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr
225 230 235 240
Phe Gln Lys Trp Ala Ser Val Val Val Pro Leu Gly Lys Glu Gln Tyr
245 250 255
Tyr Thr Cys His Val Tyr His His Gly Leu Pro Glu Pro Leu Thr Leu
260 265 270
Arg Trp Glu
275
<210> 4
<211> 840
<212> DNA
<213>mouse (Mus musculus)
<400> 4
catatgggcc cacactcgct gaggtatttc gtcaccgccg tgtcccggcc cggcctcggg 60
gagccccggt acatggaagt cggctacgtg gacgacacgg agttcgtgcg cttcgacagc 120
gacgcggaga atccgagata tgagccgcgg gcgcggtgga tggagcagga ggggcccgag 180
tattgggagc gggagacaca gaaagccaag ggcaatgagc agagtttccg agtggacctg 240
aggaccctgc tcggctacta caaccagagc aagggcggct ctcacactat tcaggtgatc 300
tctggctgtg aagtggggtc cgacgggcga ctcctccgcg ggtaccagca gtacgcctac 360
gacggctgcg attacatcgc cctgaacgaa gacctgaaaa cgtggacggc ggcggacatg 420
gcggcgctga tcaccaaaca caagtgggag caggctggtg aagcagagag actcagggcc 480
tacctggagg gcacgtgcgt ggagtggctc cgcagatacc tgaagaacgg gaacgcgacg 540
ctgctgcgca cagattcccc aaaggcccat gtgacccatc acagcagacc tgaagataaa 600
gtcaccctga ggtgctgggc cctgggcttc taccctgctg acatcaccct gacctggcag 660
ttgaatgggg aggagctgat ccaggacatg gagcttgtgg agaccaggcc tgcaggggat 720
ggaaccttcc agaagtgggc atctgtggtg gtgcctcttg ggaaggagca gtattacaca 780
tgccatgtgt accatcacgg gctgcctgag cccctcaccc tgagatggga gtaactcgag 840
<210> 5
<211> 99
<212> PRT
<213>mouse (Mus musculus)
<400> 5
Met Ile Gln Lys Thr Pro Gln Ile Gln Val Tyr Ser Arg His Pro Pro
1 5 10 15
Glu Asn Gly Lys Pro Asn Ile Leu Asn Cys Tyr Val Thr Gln Phe His
20 25 30
Pro Pro His Ile Glu Ile Gln Met Leu Lys Asn Gly Lys Lys Ile Pro
35 40 45
Lys Val Glu Met Ser Asp Met Ser Phe Ser Lys Asp Trp Ser Phe Tyr
50 55 60
Ile Leu Ala His Thr Glu Phe Thr Pro Thr Glu Thr Asp Thr Tyr Ala
65 70 75 80
Cys Arg Val Lys His Ala Ser Met Ala Glu Pro Lys Thr Val Tyr Trp
85 90 95
Asp Arg Asp
<210> 6
<211> 306
<212> DNA
<213>mouse (Mus musculus)
<400> 6
catatgatcc agaaaacccc tcaaattcaa gtatactcac gccacccacc ggagaatggg 60
aagccgaaca tactgaactg ctacgtaaca cagttccacc cgcctcacat tgaaatccaa 120
atgctgaaga acgggaaaaa aattcctaaa gtagagatgt cagatatgtc cttcagcaag 180
gactggtctt tctatatcct ggctcacact gaattcaccc ccactgagac tgatacatac 240
gcctgcagag ttaagcatgc cagtatggcc gagcccaaga ccgtctactg ggatcgagac 300
atgtaa 306
Claims (8)
1. a kind of CTL epitope polypeptide of Cryptosporidum parvum, which is characterized in that the amino acid sequence of the polypeptide such as SEQ ID
Shown in NO.1.
2. encoding the nucleic acid of the CTL epitope polypeptide of Cryptosporidum parvum described in claim 1.
3. nucleic acid according to claim 2, which is characterized in that the nucleotide sequence of the nucleic acid such as SEQ ID NO.2 institute
Show.
4. recombinant expression carrier, expression cassette, transgenic cell line, recombinant bacterium or recombination containing nucleic acid described in Claims 2 or 3
Viral vectors.
5. nucleic acid described in the CTL epitope polypeptide of Cryptosporidum parvum described in claim 1, Claims 2 or 3, claim 4 institute
It states recombinant expression carrier, expression cassette, transgenic cell line, recombinant bacterium or recombinant viral vector and is preparing Cryptosporidum parvum vaccine
In application.
6. a kind of Cryptosporidum parvum vaccine, which is characterized in that the active constituent of the Cryptosporidum parvum vaccine is following object
At least one of matter:
A. the CTL epitope polypeptide of Cryptosporidum parvum, amino acid sequence is as shown in SEQ ID NO.1；
B. the nucleic acid of the CTL epitope polypeptide of encoding amino acid sequence Cryptosporidum parvum as shown in SEQ ID NO.1；
C. nucleotide sequence nucleic acid as shown in SEQ ID NO.2；
D. recombinant expression carrier as claimed in claim 4, expression cassette, transgenic cell line, recombinant bacterium or recombinant viral vector.
7. Cryptosporidum parvum vaccine as claimed in claim 6, which is characterized in that the vaccine further includes one or more vaccines
Adjuvant.
8. nucleic acid described in the CTL epitope polypeptide of Cryptosporidum parvum described in claim 1, Claims 2 or 3, claim 4 institute
Recombinant expression carrier, expression cassette, transgenic cell line, recombinant bacterium or recombinant viral vector are stated in preparation CD8+T lymphocyte increases
Grow the application in agent.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111537715A (en) * | 2020-07-09 | 2020-08-14 | 北京维德维康生物技术有限公司 | Cryptosporidium parvum antibody detection test strip and application thereof |
| CN113304254A (en) * | 2021-06-07 | 2021-08-27 | 吉林大学 | Cryptosporidium parvum Cp15 recombinant invasive lactic acid bacteria live vector vaccine |
| CN113754749A (en) * | 2021-10-09 | 2021-12-07 | 周口师范学院 | Cryptosporidium parvum Gp40/15 protein epitope polypeptide and adenovirus vector vaccine thereof |
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| WO2007047189A2 (en) * | 2005-10-11 | 2007-04-26 | Iogenetics, Llc | Targeted biocides |
| CN101880328A (en) * | 2010-06-02 | 2010-11-10 | 中国农业科学院上海兽医研究所 | Th multi-epitope gene and fusion protein of cryptosporidium parvum and application thereof |
| CN102219858A (en) * | 2010-04-16 | 2011-10-19 | 中国农业科学院上海兽医研究所 | Cryptosporidium parvum CTL multi-epitope gene and fusion protein and application thereof |
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2019
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| WO2007047189A2 (en) * | 2005-10-11 | 2007-04-26 | Iogenetics, Llc | Targeted biocides |
| CN102219858A (en) * | 2010-04-16 | 2011-10-19 | 中国农业科学院上海兽医研究所 | Cryptosporidium parvum CTL multi-epitope gene and fusion protein and application thereof |
| CN101880328A (en) * | 2010-06-02 | 2010-11-10 | 中国农业科学院上海兽医研究所 | Th multi-epitope gene and fusion protein of cryptosporidium parvum and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111537715A (en) * | 2020-07-09 | 2020-08-14 | 北京维德维康生物技术有限公司 | Cryptosporidium parvum antibody detection test strip and application thereof |
| CN113304254A (en) * | 2021-06-07 | 2021-08-27 | 吉林大学 | Cryptosporidium parvum Cp15 recombinant invasive lactic acid bacteria live vector vaccine |
| CN113754749A (en) * | 2021-10-09 | 2021-12-07 | 周口师范学院 | Cryptosporidium parvum Gp40/15 protein epitope polypeptide and adenovirus vector vaccine thereof |
| CN113754749B (en) * | 2021-10-09 | 2024-02-09 | 周口师范学院 | Cryptosporidium parvum Gp40/15 protein epitope polypeptide and adenovirus vector vaccine thereof |
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| CN109942693B (en) | 2020-12-25 |
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