CN118146955A - Cryptosporidium parvum 23kDa mucin CP23 gene deletion insect strain and application thereof - Google Patents
Cryptosporidium parvum 23kDa mucin CP23 gene deletion insect strain and application thereof Download PDFInfo
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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
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Abstract
The invention discloses a cryptosporidium parvum 23kDa mucin CP23 gene deletion insect strain and application thereof. The invention knocks out the CP23 gene of cryptosporidium parvum IIdA G1 subtype isolate based on CRISPR/Cas9 gene editing technology, and obtains a CP23 gene deletion insect strain; researches show that the in vitro invasion and growth and development of the cryptosporidium parvum are affected after the CP23 is deleted, the gene deletion insect strain can be cultured in vitro, and the in vitro growth amount of the lotus insects is obviously reduced; after in vivo immunization, the amount of the lotus insects in the mice is also obviously reduced, the ovulation peak period is delayed, the ovulation maintenance time is shortened, and the death of the C57BL/6J mice after infection is avoided; the vaccine can reduce the infection strength of the cryptosporidium parvum to a host after the CP23 gene deletion strain is immunized, has the advantage of reduced toxicity, and can be used for preventing the cryptosporidium parvum infection and preparing the cryptosporidium parvum attenuated vaccine.
Description
Technical Field
The invention belongs to the technical field of genetic engineering. More particularly, relates to a cryptosporidium parvum 23kDa mucin CP23 gene deletion insect strain and application thereof.
Background
Cryptosporidium parvum (Cryptosporidium parvum) is an important zoonotic pathogen parasitic on the digestive tract epithelium of humans and various animals, and frequently causes moderate to severe diarrhea, and is the second biggest pathogen causing diarrhea. Cryptosporidium parvum is widely distributed in animals, humans and natural environments, and is mainly transmitted in oocyst form through a faecal route, so that severe diarrhea of children and young animals is caused, growth retardation and cognitive impairment are caused, and host death is caused when severe diarrhea is caused. The infection rate of the cryptosporidium young animals is about 70%, the transmission rate between the human and the animals is high, the human health is endangered, and meanwhile, huge economic loss is brought to the livestock and poultry raising industry.
At present, the prevention measures and treatment means for the cryptosporidiosis are very limited, and only nitazoxanide is approved by the FDA and can be used for clinical treatment of the human cryptosporidiosis. Nitazoxanide has a certain therapeutic effect on cryptosporidiosis of immunocompromised population and children over 1 year old, but has little effect on immunocompromised and organ transplanted population. However, there is no effective vaccine for preventing cryptosporidiosis, no commercial cryptosporidiosis vaccine exists in the market at present, and subunit vaccines such as DNA recombination and polypeptide have poor immune protection effect. Because of the restriction of technical bottlenecks such as genetic manipulation of Cryptosporidium, people have insufficient knowledge of invasion and growth and development mechanisms of Cryptosporidium, so that related vaccine candidate molecules are not identified until now.
The cryptosporidium parvum mucin is one of a few protein families in an insect body, wherein mucin (CP 23) with a molecular weight of 23kDa possibly participates in the adhesion host cell process of the cryptosporidium parvum, is very important for invasion and growth and development of the cryptosporidium parvum, but is limited by technical means such as gene knockout and the like, and the biological function of the cryptosporidium parvum mucin is not clear. The existing research shows that the prepared antiserum can be specifically identified by the recombinant protein through researching the cryptosporidium parvum CP23 recombinant protein, the recombinant protein has better reactivities and immunogenicity, and the CP23 protein is one of proteins mainly identified by host cell immunity, and can induce a host to generate immunoprotective antibodies (Liu Liyuan, wang Jianyong, white cloud and the like. Procaryophyllus expression and immunogenicity analysis [ J ]. Chinese veterinary science report, 2018,38 (09): 1748-1752.DOI: 10.16303/j.cnki.1005-4545.2018.09.21.); the prior studies do not reveal the function of the CP23 gene in cryptosporidium parvum, and it is not clear whether the relationship between the CP23 gene and the virulence of the insect body can be used as a vaccine target or not. There is still a lack of vaccine for preventing and treating cryptosporidiosis at present, and in order to further reveal the function and effect of cryptosporidiosis minutissima mucin on the insect body, there is a need to develop more gene-deleted insect strains or vaccines which have better immune protection effect and can be used for preventing cryptosporidiosis infection.
Disclosure of Invention
The invention overcomes the defects of the existing gene-deleted insect strain or vaccine for preventing and treating cryptosporidium infection and provides a cryptosporidium parvum 23kDa mucin CP23 gene-deleted insect strain and application thereof.
The first object of the present invention is to provide a Cryptosporidium parvum 23kDa mucin CP23 gene-deleted insect strain.
The second object of the present invention is to provide a method for constructing a Cryptosporidium parvum 23kDa mucin CP23 gene-deleted insect strain.
A third object of the present invention is to provide an application of Cryptosporidium parvum 23kDa mucin CP23 gene-deleted insect strain.
A fourth object of the invention is to provide a cryptosporidium parvum vaccine.
A fifth object of the present invention is to provide the use of Cryptosporidium parvum 23kDa mucin CP23 gene or a reagent for deleting or knocking out Cryptosporidium parvum 23kDa mucin CP23 gene.
The above object of the present invention is achieved by the following technical scheme:
The invention provides a cryptosporidium parvum 23kDa mucin CP23 gene deletion insect strain, wherein the insect strain deletes or knocks out the cryptosporidium parvum 23kDa mucin CP23 gene by a gene editing technology, and the nucleotide sequence of the CP23 gene is shown as SEQ ID NO: 1.
Further, the Cryptosporidium parvum 23kDa mucin CP23 gene is knocked out by using a CRISPR/Cas9 gene editing technology, so that a Cryptosporidium parvum CP23 gene deletion insect strain is obtained.
The invention directly knocks out the 23kDa mucin CP23 gene of Cryptosporidium parvum by CRISPR/Cas9 technology, and successfully obtains a CP23 gene-deleted insect strain; and the CP23 gene is found to be related to the virulence of the worm body for the first time, and the infection intensity and the virulence of the CP23 gene-deleted worm strain in the C57BL/6J mice are obviously reduced. The invention shows that the CP23 gene deletion insect strain grows slowly under the in-vitro growth condition by comparing the in-vitro growth condition of the marker insect strain and the deletion insect strain, the oocyst excretion strength in-vivo infection and other indexes; under the in vivo growth condition, after the C57BL/6J mice are infected with the CP23 gene-deleted insect strain, the oocyst excretion peak period is delayed and the maintenance time is shortened, and death of the infected mice is not caused, which indicates that the CP23 gene-deleted insect strain can be used for preparing the cryptosporidium parvum vaccine.
The invention provides a construction method of a cryptosporidium parvum 23kDa mucin CP23 gene deletion insect strain, which specifically comprises the following steps:
S1, using pActin:Cas9-GFP-U6: sgTK plasmid as a template, adopting an sgRNA primer of a CP23 gene, and replacing sgTK in the template plasmid with a gRNA specific to a CP23 gene target point to construct pActin:Cas9-U6: sgCP23 plasmid;
S2, constructing pActin tdTomato-Eno-Nluc-P2A-neo plasmid serving as a homologous recombination template by taking pUPRT-Actin-tdTomato-Eno-Nluc-P2A-neo-UPRT plasmid as a template and adopting a CP23 gene homologous recombination template primer;
s3, co-transfecting the pActin:Cas9-GFP-U6: sgCP 23:5323 plasmid and the homologous recombination template pActin:tdTomato-Eno:Nluc-P2A-neo plasmid into a wild cryptosporidium parvum strain, and obtaining the cryptosporidium parvum CP23 gene deletion strain through paromomycin drug screening, PCR identification and IFA experiments.
Further, in the step S1, pActin:Cr5:Cas9-GFP-U6: sgTK plasmid is used as a template, the TK target specific sgRNA is replaced by the 23kDa mucin CP23 gene target specific sgCP by primer amplification, and the vector and the sgRNA fragment are connected based on the homologous recombination principle to obtain a single SGRNA CRISPR plasmid pActin:Cr5:Cas9-U6: sgCP23.
Preferably, the sgRNA primer sequence of the CP23 gene in step S1 is shown in SEQ ID NO:2 to 4.
Further, pActin:: tdTomato-Eno:: nluc-P2A-neo homologous recombinant plasmid in step S2 is obtained by ligating the 5 'homology arm and the 3' homology arm of the 23kDa mucin gene, the complete reading frame of the expressed autored fluorescent protein tdTomato, and the luciferase reporter gene Nluc, the drug screening gene neo.
Further preferably, the genome of the wild type cryptosporidium parvum strain of the starting insect strain is used as a template, the CP23-5'UTR and the CP23-3' UTR are amplified through primers, then the tdTomato-Eno::: nluc-P2A-Neo-UPRT plasmid is amplified to obtain a tdTomato-Nluc-Neo fragment and a pUC19 vector fragment, and seamless cloning connection is carried out based on the principle of homologous recombination to construct a recombinant plasmid pActin::: tdTomato-Eno:::: nluc P2A-Neo.
Preferably, in the step S2, the primer sequence of the CP23 gene homologous recombination template is shown as SEQ ID NO:5 to 12.
Preferably, the wild type cryptosporidium parvum strain in step S3 is a cryptosporidium parvum IIdA G1 subtype strain.
The invention provides application of a CP23 gene-deleted insect strain in preparing medicines for resisting cryptosporidiosis parvulgare.
The invention provides a cryptosporidium parvum vaccine which contains the cryptosporidium parvum 23kDa mucin CP23 gene deletion insect strain.
The invention also provides application of the cryptosporidium parvum 23kDa mucin CP23 gene serving as a target spot in screening or preparing medicines for resisting cryptosporidiosis.
In addition, the research of the invention shows that the deletion of the CP23 affects the growth of the cryptosporidium parvum in vitro and seriously affects the toxicity of the cryptosporidium parvum, so that the invention provides the application of the reagent for deleting or knocking out the cryptosporidium parvum 23kDa mucin CP23 gene in constructing a CP23 gene deleted insect strain or preparing a cryptosporidium parvum vaccine.
The invention has the following beneficial effects:
The invention provides a cryptosporidium parvum 23kDa mucin CP23 gene deletion insect strain, a construction method and application thereof. The invention successfully constructs the Cryptosporidium parvum gene deletion insect strain by deleting the 23kDa mucin CP23 gene through a gene editing technology, and discovers that the CP23 gene is related to the virulence of the insect body for the first time, the infection strength and the virulence of the CP23 gene deletion insect strain in a C57BL/6J mouse body are obviously reduced, and the deletion of the CP23 influences the growth of the insect body in vitro and seriously influences the virulence of the Cryptosporidium parvum. Researches show that after the CP23 gene is deleted from the wild cryptosporidium parvum IIdA G1 subtype strain, the strain can be cultured in vitro, the in vitro growth amount of the strain is obviously reduced, the in vivo amount of the strain is also obviously reduced, the oocyst excretion strength is obviously weakened, the death of a C57BL/6J mouse after infection can not be caused, the strain has the advantage of obviously weakened toxicity, and the strain can be used for preparing cryptosporidium parvum attenuated vaccines so as to be used for preventing cryptosporidium parvum infection.
Drawings
FIG. 1 is a schematic representation of the construction of a Δc23 insect strain.
FIG. 2 is an agarose gel electrophoresis of PCR identification of ΔcP23 insect strains.
FIG. 3 is a graph showing the results of evaluating in vitro growth replication of Δcp23 insect strains in an in vitro culture system.
FIG. 4 is a result of in vivo infection experiments evaluating the intensity of infection of Δcp23 strain in mice.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The pACT1:: cas9-GFP-U6:: sgTK plasmid, pUPRT-3HA-Nluc-P2A-neo plasmid used in the examples below were all from the university of Washington L.David Sibley laboratory.
The wild cryptosporidium parvum IIdA G1 subtype isolate is obtained by collecting and typing and identifying a certain dairy farm in Heilongjiang, and the typing and identifying method refers to ：Li N,Zhao W,Song S,Ye H,Chu W,Guo Y,et al.Diarrhoea outbreak caused by coinfections of Cryptosporidium parvum subtype IIdA20G1 and rotavirus in pre-weaned dairy calves.Transbound Emerg Dis.2022;69:e1606-17., for self-subculturing and seed preservation in the laboratory to date.
EXAMPLE 1 construction of Cryptosporidium parvum Δcp23 strain
1. Insect-producing strain
The starting strain used in this example is a wild type Cryptosporidium parvum IIdA G1 subtype isolate of Cryptosporidium of Cluster, which has a 23kDa mucin (CP 23) gene with a nucleotide sequence as shown in SEQ ID NO: 1.
2. Construction of CRISPR knockout plasmid pActin:: cas9-U6:: sgCP23
(1) Designing a CP23 targeting site by using a gRNA online design website (http:// gRNA. Ctegd. Uga. Edu /), wherein the selected CP23 gRNA sequence is 5'-CTGCTGAACCTGCTCCACAGC-3', PAM and the selected sequence is GGG; the designed target sequences are shown in table 1 below;
table 1 construction of primers used for the Single sgRNA plasmid pActin:: cas9-U6:: sgCP23
Cas9-U6: sgCP is taken as a knockout plasmid in a CRISPR/Cas9 system, pActin:Cas9-GFP-U6: sgTK is taken as a template, sgTK is replaced by sgCP, phanta high-fidelity enzyme (Phanta Super-FIDELITY DNA Polymerase, P501-d 1) of Novain company is used for fragment amplification, seamless cloning ligase (Uniclone One StepSeamless Cloning Kit, SC 612) of Jinsha company is used for connection to construct a single sgRNA plasmid pActin:Cas9-U6: sgCP23, and plasmids which do not contain endotoxin are extracted after sequencing identification of correct strains are subjected to amplification culture and are stored at minus 20 ℃ for standby.
(2) Construction of CP23 specific Single GRNA CRISPR plasmid
CP23 gRNA sequences were designed using EuPaGDT (http:// gRNA. Ctegd. Uga. Edu /) online tool. In the invention, the gRNA sequence is 5'-CTCTGAGTGTACAACTGCTA-3', PAM, the sequence is AGG, pACT1: cas9-GFP-U6: sgTK plasmid (purchased from http:// www.addgene.org) is used as a template for PCR amplification, and the reaction system and the reaction conditions are shown in the following tables 2 and 3;
TABLE 2PCR reaction System
TABLE 3PCR reaction conditions
After the completion of PCR, the plasmid template remaining in the PCR product was digested with Takara restriction endonuclease Dpn I, and the reaction system was as shown in Table 4 below, and the PCR tube was subjected to instantaneous centrifugation, heat treatment at 70℃for 15min and digestion at 37℃for 30min, to thereby carry out a PCR digestion reaction;
TABLE 4Dpn I digestion System
Use full gold PCR product purification kitPCR Purification Kit, EP 101-01) recovering digested PCR products, measuring the concentration by using a nucleic acid protein meter (NanoDrop 2000) of the United states Sesamer, connecting by using a seamless Cloning ligase (Uniclone One STEP SEAMLESS Cloning Kit, SC 612) of the Jinsha company, measuring the concentrations of a linearization vector and an insert (a primer with 59bp length and containing a homologous sequence) respectively as shown in the following Table 5, mixing 200ng (X. Mu.L) of the linearization vector and 10ng (Y. Mu.L) of the insert uniformly, adding 2X Uniclone Seamless Cloning Mix, and connecting for 1h at 50 ℃;
table 5 seamless cloning ligation System
50 Mu L of the ligation product is transformed into DH5 alpha competent cells, coated on LB/Amp solid medium and cultured in a 37 ℃ incubator for 10-12h; picking a single colony, placing the single colony in 1mL of LB/Amp liquid culture medium, and carrying out shaking culture at 37 ℃/180rpm until bacterial liquid is turbid; taking 500 μl of bacterial liquid, and delivering to Optimaceae sequencing by the company limited biotechnology company, extracting plasmids after amplifying and culturing bacterial liquid with correct sequencing, and the constructed plasmids are named as follows: pActin: cas9-U6:: sgCP23.
3. PActin construction of tdTomato-Eno:: nluc-P2A-neo homology template
(1) Extraction of Cryptosporidium parvum oocyst DNA
Oocysts of the cryptosporidium parvum IIdA G1 subtype isolate were extracted into cryptosporidium parvum oocyst DNA according to the DNA extraction Kit of kajie company, germany (dnase Blood & Tissue Kit, 69504) instructions.
(2) PCR amplification of homologous recombination sequences
The homologous recombinant plasmid pActin was constructed by using the prepared cryptosporidium parvum oocyst DNA and the plasmid UPRT-tdTomato-Nluc-P2A-neo-UPRT as templates, tdTomato-Eno:::: nluc-P2A-neo, replacing the 5'UTR and 3' UTR of the UPRT gene on the template plasmid with the 5'UTR and 3' UTR of CP23, amplifying the 5'UTR and 3' UTR sequences of CP23 of 100bp or more from cryptosporidium parvum oocyst DNA using the primer sequences as shown in Table 6 below, and then amplifying the Actin-tdTomato-Nluc-P2A-neo-UP and the plasmid vector fragment from the UPRT-tdTomato-N2A-neo-UPRT plasmid, and the specific amplification reaction system and reaction procedure are shown in tables 2 and 3.
TABLE 6pActin tdTomato-Eno:: nluc-P2A-neo construction of plasmid primers
(3) Recovery of fragments of interest
The PCR results were detected by 1% agarose gel electrophoresis, the target fragment was recovered using DNA gel recovery kit (FastPure Gel DNA Extraction Mini Kit, DC 301-01) from Nanjinopran, according to the kit protocol, and the concentration of the gel recovered fragment was detected by a nucleic acid protein concentration meter (Nanodrop 2000).
(4) PActin construction of tdTomato-Eno:: nluc-P2A-neo homologous template plasmid
The ligation reaction system was prepared as shown in Table 5 above, by following the one-step method of the Kit for seamless Cloning (Uniclone One STEP SEAMLESS Cloning Kit, SC 612) instructions of Beijing Jinsha. The longest fragment was used as a plasmid vector, and ng was used as the optimum amount according to (0.02×base number); the remaining fragments were used as inserts, and ng was used as the optimum amount according to (0.04 Xthe number of bases). Mixing uniformly, performing instantaneous centrifugation, connecting for 1h at 50 ℃, transferring into DH5 alpha competent cells, performing amplification culture on the strain with correct sequencing identification, extracting endotoxin-free plasmid pActin tdTomato-Eno Nluc-P2A-neo, and preserving at-20 ℃ for later use.
4. Construction of Cryptosporidium parvum Gene-deleted insect Strain Deltacp 23
(1) Treatment of Cryptosporidium parvum IIdA G1 subtype isolates oocysts
Collecting positive mouse feces of Cryptosporidium parvum IIdA G1 subtype insect strain, storing in 5% potassium dichromate solution, vortex oscillating the feces suspension to homogeneous state, and sieving with 20 mesh and 60 mesh sieve to remove residual padding and impurities.
Filtering with a screen: the homogenized fecal solution is sequentially screened through a 20-mesh screen and a 60-mesh screen to remove residual padding and impurities, and the residue on the screen is washed with pre-cooled pure water without forced extrusion during screening. After the fecal filtrate had settled naturally for 10min, significant delamination occurred, the supernatant (bottom sediment) was poured into a 500mL centrifuge bottle, centrifuged at 4000r/min for 10min, and the supernatant carefully discarded. The steps were repeated, and the pellet was resuspended in 80mL of purified water and the centrifuge flask was vigorously shaken to ensure adequate dispersion of the pellet to form a homogeneous suspension.
Sucrose density gradient centrifugation: using saturated sucrose solutions to prepare 1:2 and 1:4 gradient sucrose solutions, 20mL of 1:4 sucrose solution was added to each 50mL centrifuge tube, then 20mL of 1:2 sucrose solution was slowly injected from the bottom using a syringe, significant delamination was observed at the 20mL scale of the centrifuge tube, then the upper step suspension was slowly added along the sucrose solution level (to reduce the impact force, the head of the pasteur tube was cut off), and centrifugation was performed at 1000 Xg for 25min with a lift centrifuge acceleration adjusted to a minimum of 1.
Crude oocyst fluid was obtained: after centrifugation, the top 20mL of the solution is sucked by a Pasteur pipe, then the oocyst zone solution between 10-30mL of scales of the centrifuge tube is sucked into the centrifuge tube, pre-cooled pure water is added into the centrifuge tube according to the ratio of 1:1, the centrifugation is carried out for 10min at 4000r/min, and 2mL of pre-cooled pure water is used for re-suspension to obtain crude oocyst solution.
Cesium chloride density gradient centrifugation: a cesium chloride solution was prepared from 34.08mL of water+7.25 g of cesium chloride, 1mL of cesium chloride solution was added to each 1.5mL low adsorption EP tube, the tip of the pipette tip was cut off, 500. Mu.L of crude oocyst liquid was carefully added to the upper layer of cesium chloride solution along the liquid surface with a pipette, and centrifuged at 13200r/min for 3min.
Oocyst enrichment: after centrifugation, a distinct layered oocyst band appears at the 1mL scale of the centrifuge tube, each tube is aspirated into a new 1.5mL low adsorption EP tube at a 1:1 proportion of pre-cooled pure water, 13200r/min and 3min.
Oocyst count and preservation: the supernatant was thoroughly discarded, each tube of pellet was resuspended with 1mL of pre-chilled PBS total volume, the oocyst fluid was enriched into a 1.5mL low-adsorption EP tube, 10. Mu.L of oocyst fluid was counted in a hemocytometer after pipetting well, and finally 10. Mu.L of tri-antibody (penicillin, streptomycin, and amphotericin B mixed antibiotic solution, which inhibited bacterial and fungal growth in oocyst fluid) was added and stored in a freezer at 4 ℃. The purified oocysts are generally sub-packed in a plurality of 1.5mL low adsorption EP tubes according to the quantity of 1X 10 7 oocysts, so that the phenomenon that the oocysts are excessively high in quantity and are influenced by gravity to form compact oocyst precipitates to influence the activity of the oocysts is avoided.
(2) Acquisition of Cryptosporidium parvum IIdA G1 subtype isolate sporozoites
1.5X10 7 oocysts were aspirated and placed on an ice box, 200. Mu.L of sodium hypochlorite stock solution and 600. Mu.L of PBS were added and the ice bath was performed for 10min. Centrifugation was performed at 13200rpm at 4℃for 3min, the supernatant was discarded thoroughly, and the oocysts were washed 3 times with PBS. Afterwards, the treated oocysts were resuspended with 400. Mu.L of 1% BSA, then 400. Mu.L of 1.5% sodium taurocholate was added to give a final concentration of 0.75% sodium taurocholate, and the mixture was placed in a 37℃water bath for incubation for 60-70min. When the decoy rate exceeded 80%, the supernatant was discarded by centrifugation at 13200rpm for 3min at room temperature, sporozoites were resuspended in 1mL of PBS at room temperature, and the supernatant was discarded by centrifugation at 13200rpm for 3min, and repeated 3 times.
(3) Preparation of transfection System and electric transfection
The supernatant was discarded from the suspension of the previous step, and sporozoites were resuspended after preparing the electrotransfection solution with the Lonza 4D-nucleic acid TM X Kit, V4XC-2024 Kit, the electrotransfection solution preparation being shown in Table 7, the total system being 100. Mu.L;
TABLE 7 electrotransfection System
The electrotransfer buffer consists of 65.6 mu L SF buffer and 14.4 mu L S1 buffer, and the prepared CRISPR direct knockout plasmid and homologous repair plasmid are both 50 mu g, so that the plasmid concentration is adjusted to 5000 ng/. Mu.L after plasmid extraction.
(4) Neutralizing gastric acid and lavage infection
1 GKO mice (purchased from the institute of medical laboratory animals at the national academy of medical sciences and bred at the laboratory animal center at the agricultural university of south China) of 3 to 5 weeks old were prepared in advance, 200. Mu.L of 8% saturated sodium bicarbonate solution was administered to GKO mice of 3 to 5 weeks old to neutralize the stomach acid of the mice, and after 5 minutes of reaction, 100. Mu.L of the solution containing sporozoites was infused into the stomach of the mice. 24h after stomach irrigation, the common drinking water of GKO mice is replaced by 16g/L paromomycin sulfate solution, and the screening of the edited insect strains is carried out under the drug selection pressure.
(5) Luciferase value detection
Infection of HCT-8 cells (purchased from cell bank of the national academy of sciences, CBP 60030) with a small amount of sporozoites remaining from the previous step, after incubation for 24h, by usingThe Luciferase ASSAY SYSTEM kit (Promega corporation, nano-Glo Luciferase kit, N1120, U.S.A.) detects the relative expression level of Nluc Nano-Luciferase to determine if electrotransformation was successful.
The ovulation status of Cryptosporidium parvum was subsequently monitored by detecting luciferase expression in the faeces of infected mice. One mouse fresh feces was collected 5 days, 10 days and 15 days after infection, placed in a 1.5mL centrifuge tube, 103 mm glass beads and 1mL feces lysate were added, broken with shaking for 1min, and then centrifuged at 19000g for 1min. 50. Mu.L of the supernatant was added to a 96-well assay plate, and the relative expression level of Nluc nanoluciferase was then assayed using the above-described kit. When the mouse fecal luciferase reached 10 ten thousand, the mouse feces were collected daily and oocysts were purified every three days.
(6) Identification of Δcp23 insect strains
Identifying the gene editing insect strain through PCR1, PCR2 and PCR3 amplification reaction, wherein primers are shown in table 8, and a PCR reaction system and a reaction program are shown in tables 2 and 3;
table 8 Deltacp 23 insect strain PCR identification primer
Schematic diagram 1 of Δcp23 strain construction shows that PCR1 and PCR2 band in the results indicate that tdTomato, luciferase fragment Nluc and drug screening fragment Neo have been integrated into cryptosporidium parvum CP23 site; PCR3 detects whether gRNA targets the CP23 locus of the Cryptosporidium parvum genome, if the wild Cryptosporidium parvum of the control group has a PCR3 band, and the experimental group has no PCR3 band, the CP23 gene is successfully knocked out. The identification result is shown in fig. 2, which shows that the delta cp23 insect strain is successfully constructed.
Example 2 in vitro growth experiments of Cryptosporidium parvum Δcp23 strain
In this example, the growth status of the gene editing insect strain was evaluated by an in vitro growth experiment. Measuring the relative expression quantity of luciferase by using different gradient quantity gene editing insect strains, and quantifying the insect body quantity by measuring the luciferase value if the luciferase value and the gene editing insect strain quantity show positive correlation linear relation; and comparing the luciferase values of the cell cultures at different time points after the infection of the genome marker group and the genome deletion group insect body, and further estimating the copy and proliferation condition of the gene editing insect strain. If the gene deletion has an effect on the proliferation of the insect bodies, and the statistical difference of the values of the two groups of luciferases is analyzed at corresponding time points, the situation that the proliferation of the insect bodies of the gene deletion group is slow occurs.
In vitro culturing of gene editing insect strains: oocysts were taken to infect HCT-8 cells: experiments were performed on 48-well cell culture plates and when the confluence of HCT-8 cells reached more than 80%, 1.0X10 5 oocysts were infected per well, as follows:
The gene marker group (CP 23-N-3HA: the CP23-N-3HA insect strain obtained by inserting 3 XHA after the 5 th amino acid of the CP23 protein by CRISPR/Cas9 gene editing technology, the preparation method is the same as that of delta CP 23) and the gene deletion group (delta CP 23) are respectively obtained by putting 2.5X10 6 oocysts into a 1.5mL low-adsorption centrifuge tube, adding 200 mu L sodium hypochlorite mother liquor and 600 mu L PBS, and carrying out ice bath for 10min; centrifuging at 13200rpm at 4deg.C for 3min, discarding supernatant thoroughly, washing oocysts with PBS for 3 times; the original HCT-8 cell culture medium in the cell holes is thoroughly discarded, new 2% FBS1640 culture medium is replaced to resuspend the oocysts, and finally, an equal volume of oocyst-culture medium is added into each HCT-8 cell hole.
The luciferase values of each well were measured at 3, 12, 24, 36, 48h after infection, four biological replicates were set at each time point, the relative expression levels of luciferase were detected, and the results were counted.
The CP23-N-3HA control group was used to detect the change in proliferation of the delta CP23 strain infected with HCT-8 cells at different time points, and the result is shown in fig. 3, wherein the proliferation value of the delta CP23 group is significantly lower than that of the CP23-N-3HA control group at 3h after infection, and the difference is significant (p=0.0029); and also showed significant differences (p=0.0001) at time points of 12h, 24h, 36h and 48 h. The above results demonstrate that CP23 deficiency affects cryptosporidium parvum in vitro invasion and growth.
EXAMPLE 3 in vivo experiments of Cryptosporidium parvum Δcp23 strain
To investigate the effect of CP23 protein on the growth and reproduction of cryptosporidium parvum in vivo, C57BL/6J immunized normal mice (derived from the experimental animal center in guangdong province) were infected with CP23-N-3HA marker strain and Δcp23 knockout strain, respectively, and the oocyst excretion intensity comparison and pathogenicity difference were compared, as follows:
(1) Grouping: 3-week-old C57BL/6J mice were randomly divided into three groups according to weight average and sex, 5 mice per infected group, 10 5 oocysts per mouse were infected, and a negative control group was set as a non-infected group, 3 mice per mouse were housed in separate cages.
(2) Infection: after each group of mice is subjected to single-cage adaptive feeding for three days, 105 oocysts are filled in the stomach of each mouse, the feces of each mouse are collected individually and completely, the feces of the mice with different gene editing insect strains are collected, the gloves are replaced, pollution is prevented, and records are made.
(3) The mouse manure nano-luciferase assay was performed in the same manner as in example 1 by assaying the mouse manure luciferase values to evaluate the effect on cryptosporidium parvum infectivity and pathogenicity after CP23 deletion.
(4) Weighing the mice: each mouse weight change was weighed every two days, recorded, the data were statistically processed using SPSS 2346 software, and one-way analysis of variance was performed to determine if there was a significant difference in mouse weight change between the gene marker set and the gene deletion knockout set.
Results as shown in figure 4, the CP23-N-3HA positive control mice entered the peak oocyst excretion period on day 4 post infection, which may last for 6 days; whereas the Δcp23 group mice entered peak at day 8 post-infection, with a duration of 2 days. There was no significant difference in luciferase values between the CP23-N-3HA group and the Δcp23 group mouse faeces at day 14 post infection. The result shows that the deletion of CP23 delays the replication and proliferation of the insect strain in mice, shortens the duration of peak period, reduces infectivity, does not cause death of the mice, and can be used for preparing the cryptosporidium parvum vaccine as a vaccine candidate insect strain of the cryptosporidium parvum.
In summary, the invention provides a Cryptosporidium parvum CP23 gene deleted strain (delta CP 23) obtained based on CRISPR/Cas9 gene editing technology and Cryptosporidium parvum IIdA G1 subtype isolate; researches show that the in vitro invasion and growth and development of the cryptosporidium parvum are affected after the CP23 is deleted, the delta CP23 strain can be cultured in vitro, the in vitro growth lotus is obviously reduced, the in vitro lotus is also obviously reduced in mice, the ovulation peak period is delayed, the ovulation maintenance time is shortened, the death of the C57BL/6J mice after infection is avoided, the growth and development of the cryptosporidium parvum can be affected after the CP23 gene is deleted, the infection intensity of the cryptosporidium parvum to a host is relieved, and the advantages of reduced toxicity are achieved, and the cryptosporidium parvum attenuated vaccine can be used for preparing the cryptosporidium parvum attenuated vaccine.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. The cryptosporidium parvum 23kDa mucin CP23 gene deletion insect strain is characterized in that the insect strain deletes or knocks out the cryptosporidium parvum 23kDa mucin CP23 gene by a gene editing technology, and the nucleotide sequence of the CP23 gene is shown as SEQ ID NO: 1.
2. The method for constructing a gene-deleted insect strain of claim 1, wherein the cryptosporidium parvum CP23 gene-deleted insect strain is obtained by knocking out the cryptosporidium parvum 23kDa mucin CP23 gene using CRISPR/Cas9 gene editing technology.
3. The construction method according to claim 2, characterized in that it comprises the following steps:
S1, using pActin:Cas9-GFP-U6: sgTK plasmid as a template, adopting an sgRNA primer of a CP23 gene, and replacing sgTK in the template plasmid with a gRNA specific to a CP23 gene target point to construct pActin:Cas9-U6: sgCP23 plasmid;
S2, constructing pActin tdTomato-Eno-Nluc-P2A-neo plasmid serving as a homologous recombination template by taking pUPRT-Actin-tdTomato-Eno-Nluc-P2A-neo-UPRT plasmid as a template and adopting a CP23 gene homologous recombination template primer;
s3, co-transfecting the pActin:Cas9-GFP-U6: sgCP 23:5323 plasmid and the homologous recombination template pActin:tdTomato-Eno:Nluc-P2A-neo plasmid into a wild cryptosporidium parvum strain, and obtaining the cryptosporidium parvum CP23 gene deletion strain through paromomycin drug screening, PCR identification and IFA experiments.
4. The construction method according to claim 3, wherein the sgRNA primer sequence of the CP23 gene in step S1 is shown in SEQ ID NO:2 to 4.
5. The construction method according to claim 3, wherein the primer sequence of the CP23 gene homologous recombination template in step S2 is shown in SEQ ID NO:5 to 12.
6. The method according to claim 3, wherein the wild-type Cryptosporidium parvum strain in step S3 is a Cryptosporidium parvum IIdA G1 subtype strain.
7. The use of the CP23 gene-deleted strain of claim 1 in the preparation of a medicament against cryptosporidiosis parvulgare.
8. A cryptosporidium parvum vaccine comprising the cryptosporidium parvum CP23 gene-deleted strain of claim 1.
9. The application of cryptosporidium parvum 23kDa mucin CP23 gene as a target spot in screening or preparing medicines for resisting cryptosporidiosis is characterized in that the nucleotide sequence of the CP23 gene is shown as SEQ ID NO: 1.
10. The application of a reagent for deleting or knocking out cryptosporidium parvum 23kDa mucin CP23 gene in constructing a CP23 gene deleted insect strain or preparing a cryptosporidium parvum vaccine is characterized in that the nucleotide sequence of the CP23 gene is shown as SEQ ID NO: 1.
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