Cryptosporidium parvum dhr1 gene-replaced strain, construction method and application thereof

Abstract

The application discloses a Cryptosporidium parvum CpDHR1 gene replacement strain and a construction method and application thereof. The application researches and finds that the CpDHR1 gene of Cryptosporidium parvum is related to virulence, the CpDHR1 gene of a high-virulence strain is replaced by the CpDHR1 gene of a low-virulence strain through a gene editing technology, and a Cryptosporidium parvum CpDHR1 gene replacement strain is successfully constructed. Research shows that after the CpDHR1 gene of a high-virulence wild-type Cryptosporidium parvum IIdA20G1 subtype strain is replaced, the strain can still be cultured in vitro, but the in-vitro growth of the strain is significantly reduced, the virulence of the strain is weakened, the oocyst excretion intensity of the strain is weakened, the oocyst load in mice is reduced, the clinical symptoms of the mice are alleviated, the survival time of the mice is prolonged, the virulence of the strain is significantly weakened, the strain can be used for preparing a Cryptosporidium parvum low-virulence vaccine product, and Cryptosporidium parvum infection can be better prevented.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and genetic engineering, and more specifically, to a Cryptosporidium microsporidium CpDHR1 gene replacement strain, its construction method, and its application. Background Technology

[0002] Cryptosporidium microsporum ( Cryptosporidium parvum Cryptosporidia is a highly dangerous zoonotic parasite. As one of the two main dominant species infecting humans, it is widely present in farmed animals such as cattle, horses, and sheep. Among the four common Cryptosporidia species that infect cattle, *Cryptospora parvum* is highly pathogenic to pre-weaned calves. *Cryptospora parvum* is mainly transmitted via the fecal-oral route, parasitizing the intestinal mucosal epithelial cells of the host. Infection causes cryptosporidiosis, characterized by gastrointestinal dysfunction and watery diarrhea; severe cases can lead to death. Cryptosporidia not only causes significant economic losses to livestock farming but also poses a persistent threat to public health.

[0003] Cryptosporidium parvum vaccines are currently still in the research stage, and no commercially available vaccines have been launched. The main research and development technologies include two categories: live attenuated vaccines and live vector subunit vaccines. Significant progress has been made in the research of related gene attenuation targets. For example, existing research has publicly used CRISPR / Cas9 technology to knock out key virulence genes (such as GP60), significantly reducing the in vitro growth ability and in vivo infectivity of the strains, thus significantly weakening their virulence and mimicking natural infection to elicit strong immunity. However, because Cryptosporidium is difficult to passage continuously in vitro and undergo reverse genetic manipulation, its genetic manipulation technology has long been lagging behind, resulting in insufficient understanding of the invasion and development mechanisms of Cryptosporidium. Therefore, there are currently few gene-edited strains that can be used for attenuated vaccine preparation. At present, vaccines and genetic engineering interventions for Cryptosporidium parvum infection remain extremely limited. In-depth understanding of the key molecular differences between highly virulent and low-virulence strains is of significant scientific and application value for developing new prevention and control strategies. Therefore, this invention application is proposed. Summary of the Invention

[0004] The technical problem to be solved by this invention is to overcome the shortcomings of existing gene-edited strains or vaccines for the prevention and control of Cryptosporidium infection. By systematically analyzing the genetic basis and molecular mechanism of Cryptosporidium virulence, this invention provides a Cryptosporidium microsporidium CpDHR1 gene replacement strain, its construction method and application.

[0005] The first objective of this invention is to provide a Cryptosporidium microsporidium CpDHR1 gene replacement strain.

[0006] The second objective of this invention is to provide a method for constructing a Cryptosporidium microsporidium CpDHR1 gene replacement strain.

[0007] A third objective of this invention is to provide an application of Cryptosporidium microsporidium CpDHR1 gene replacement strains.

[0008] The fourth objective of this invention is to provide a product containing a Cryptosporidium microsporidium CpDHR1 gene replacement strain.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] This invention provides a Cryptosporidium microsporidium CpDHR1 gene-replaced strain. The CpDHR1 gene of a highly virulent Cryptosporidium microsporidium strain is replaced with the CpDHR1 gene of a low-virulence strain using CRISPR / Cas9 gene editing technology, thereby obtaining a Cryptosporidium microsporidium CpDHR1 gene-replaced strain. The nucleotide sequence of the CpDHR1 gene of the highly virulent strain is shown in SEQ ID NO: 1; the nucleotide sequence of the CpDHR1 gene of the low-virulence strain is shown in SEQ ID NO: 2.

[0011] In the early stages of this invention, genetic hybridization and identification analysis were performed on two Cryptosporidium microsporidium isolates with different virulence. It was found that the Cryptosporidium DEAH / RHA RNA helicase DHR1 (CpDHR1 gene) was identified as a potential virulence-related gene in both high polymorphism screening and positive selection analysis. Base differences were found between Cryptosporidium microsporidium isolates with different virulence. The nucleotide sequence of the CpDHR1 gene in the highly virulent IIdA20G1 subtype Cryptosporidium strain is shown in SEQ ID NO: 1; the nucleotide sequence of the CpDHR1 gene in the low-virulence IIdA19G1 subtype strain is shown in SEQ ID NO: 2, with the gene number cgd5_4090.

[0012] This invention utilizes gene editing technology to replace the CpDHR1 gene of a highly virulent Cryptosporidium strain with that of a less virulent strain, successfully constructing a Cryptosporidium c. microsporidium strain with the replaced CpDHR1 gene. Studies show that after replacing the CpDHR1 gene in the highly virulent wild-type Cryptosporidium IIdA20G1 subtype strain, it can still be cultured in vitro, but the in vitro parasite load is significantly reduced, virulence is weakened, and the parasite load in mice is also reduced, with weakened oocyst excretion. Simultaneously, it alleviates clinical symptoms in mice and prolongs survival time, demonstrating the advantage of significantly reduced virulence. This strain can be used to prepare a Cryptosporidium c. microsporidium attenuated vaccine for the prevention of Cryptosporidium infection. Based on the crucial role of CpDHR1 in the pathogenesis of Cryptosporidium and the stable attenuation effect caused by its replacement, it can serve as a new candidate strain for attenuated vaccine products for Cryptosporidium c. microsporidium.

[0013] Preferably, the Cryptosporidium microsporum refers to ΔCpDHR1-HLJ :CpDHR1-GD strain.

[0014] More preferably, the highly toxic insect strain refers to the highly toxic IIdA20G1 subtype insect strain; the low-toxic insect strain refers to the low-toxic IIdA19G1 subtype insect strain.

[0015] This invention provides a method for preparing a CpDHR1 gene-replaced insect strain, comprising the following steps: S1. Using pAct::Cas9-GFP-U6::sgTK plasmid as template, and employing sgRNA primers of the low-virulence insect strain CpDHR1 gene, a double sgRNA CRISPR plasmid pAct::Cas9-U6::sg CpDHR1-1-U6::sg CpDHR1-2 was constructed. S2. Using the plasmid cgd8_5420-Nluc-P2A-neo-mCh-cgd8_5420 and the gDNA of the wild-type Cryptosporidium microsporum strain as templates, and using the homologous recombination template primers of the low-virulence strain CpDHR1 gene, the homologous recombination plasmid CpDHR1-Nluc-P2A-neo-mCh-CpDHR1 was constructed. S3. The pAct::Cas9-U6::sg CpDHR1-1-U6::sg CpDHR1-2 plasmid and the CpDHR1-Nluc-P2A-neo-mCh-CpDHR1 homologous recombinant plasmid were co-transfected into wild-type Cryptosporidium microsporum strains. The Cryptosporidium microsporum CpDHR1 gene replacement strains were obtained by drug screening and PCR identification.

[0016] Preferably, the primer sequences for the sgRNA of the low-virulence strain CpDHR1 in step S1 are shown in SEQ ID NO: 3~10.

[0017] Preferably, the primer sequences for homologous recombination of the low-virulence insect strain CpDHR1 gene in step S2 are shown in SEQ ID NO: 11~20.

[0018] Preferably, the wild-type Cryptosporidium microsporidium strain in step S3 is the Cryptosporidium microsporidium IIdA20G1 subtype strain.

[0019] As a preferred embodiment, the present invention provides a more specific method for constructing a Cryptosporidium microsporidium CpDHR1 gene replacement strain: S1. Construction of the dual sgRNA CRISPR plasmid pAct::Cas9-U6::sgCpDHR1-1-U6::sgCpDHR1-2: Using pAct::Cas9-GFP-U6::sgTK plasmid as a template, the original TK gene-specific sgRNA sequence was replaced with the first target-specific sgRNA segment targeting the CpDHR1 gene (sgCpDHR1-1) via PCR amplification and homologous recombination. The constructed product was transformed into DH5α competent cells, and the intermediate plasmid pAct::Cas9-U6::sgCpDHR1-1 was obtained after screening and verification. Using the same strategy, another intermediate plasmid, pAct::Cas9-U6::sgCpDHR1-2, was constructed. Then, using the CpDHR1 CRISPR1 plasmid as a template, the plasmid backbone was amplified with primers; subsequently, using the CpDHR1 CRISPR2 plasmid as a template, the sgRNA fragment was amplified, and finally the plasmid pAct::Cas9-U6::sgCpDHR1-1-U6::sgCpDHR1-2 was constructed.

[0020] Construction of the S2.CpDHR1-Nluc-P2A-neo-mCh-CpDHR1 homologous recombinant plasmid: Using the genome of Cryptosporidium microlucum subtype IIdA20G1 as a template, the upstream homologous arm (5' UTR) and downstream homologous arm (3' UTR) of the CpDHR1 gene were amplified by PCR. Using the genome of Cryptosporidium microlucum subtype IIdA19G1 as a template, the CDS region of the CpDHR1 gene was amplified by PCR. Simultaneously, the Nluc luciferase reporter gene and the Neo resistance selection marker fragment were amplified from the plasmid carrying the Nluc and Neo genes; the mCherry red fluorescent protein coding sequence was amplified from the plasmid containing mCherry.

[0021] The four functional fragments (5'UTR, Nluc-P2A-neo-mCh, 3'UTR) obtained above, along with the PCR-linearized pUC19 vector backbone, were assembled in a one-step process using a multi-fragment homologous recombinase ligation system to finally construct the homologous recombinant plasmid CpDHR1-Nluc-P2A-neo-mCh-CpDHR1.

[0022] S3. Obtaining the CpDHR1 gene-replaced strain of Cryptosporidium microlucum: The sporozoite suspension after excoagulase treatment was mixed with the double sgRNA CRISPR plasmid pAct::Cas9-U6::sgCpDHR1-1-U6::sgCpDHR1-2 constructed in step S1 and the homologous recombination donor plasmid CpDHR1-Nluc-P2A-neo-mCh-CpDHR1 constructed in step S2. The mixture was introduced into wild-type Cryptosporidium microlucum using electroporation. After subsequent screening and verification, the CpDHR1 gene-replaced strain was finally obtained.

[0023] This invention provides a formulation for replacing the CpDHR1 gene of Cryptosporidium microsporidium for use in constructing Cryptosporidium microsporidium CpDHR1 gene replacement strains or in the preparation of Cryptosporidium microsporidium vaccines.

[0024] This invention provides the application of a formulation that replaces the CpDHR1 gene of Cryptosporidium microsporidium in the preparation of products that inhibit the growth of Cryptosporidium microsporidium.

[0025] Preferably, the formulation replaces the CpDHR1 gene of the highly virulent Cryptosporidium microsporidium strain with the CpDHR1 gene of the low-virulent strain.

[0026] Preferably, the preparation for replacing the CpDHR1 gene of Cryptosporidium microsporum is a recombinant expression vector of the CpDHR1 gene of a low-virulence strain.

[0027] This invention provides the application of knockout or replacement agents targeting the CpDHR1 gene of highly virulent Cryptosporidium microsporidium strain in the preparation of drugs for preventing Cryptosporidium microsporidium infection.

[0028] This invention also provides the application of Cryptosporidium microsporidium CpDHR1 gene replacement strains in the preparation of Cryptosporidium microsporidium attenuated products.

[0029] Preferably, the attenuated product refers to an attenuated vaccine.

[0030] This invention provides an attenuated Cryptosporidium microsporidium product containing the aforementioned Cryptosporidium microsporidium CpDHR1 gene-replaced strain.

[0031] The present invention has the following beneficial effects: This invention provides a Cryptosporidium microsporidium CpDHR1 gene-replaced strain, its construction method, and its applications. By using gene editing technology, the CpDHR1 gene in the highly virulent strain IIdA20G1 is replaced with the CpDHR1 gene from the low-virulence strain IIdA19G1, successfully constructing a Cryptosporidium microsporidium CpDHR1 gene-replaced strain. This invention is the first to discover that the CpDHR1 gene is related to the virulence of the parasite. The CpDHR1-replaced strain exhibits an attenuated phenotype in an IFN-γ knockout mouse model: its in vivo infectivity and pathogenicity are reduced, oocyst excretion is decreased, and the survival time of infected mice is prolonged. Replacing the CpDHR1 gene in the wild-type Cryptosporidium microsporidium IIdA20G1 subtype strain has the advantage of significantly reduced virulence, which can be used to prepare an attenuated Cryptosporidium microsporidium vaccine for the prevention of Cryptosporidium microsporidium infection, providing a new theoretical basis and technical support for the prevention and control of Cryptosporidium microsporidiasis. Attached Figure Description

[0032] Figure 1 This is a schematic diagram illustrating the construction principle of the CpDHR1 replacement insect strain.

[0033] Figure 2 This is the PCR identification result of the CpDHR1 replacement strain.

[0034] Figure 3 These are the sequencing analysis results of the CpDHR1 replacement strain.

[0035] Figure 4 This is an analysis of the in vitro growth ability of CpDHR1 replacement insect strains.

[0036] Figure 5 This represents the amount of oocysts excreted in mice infected with the CpDHR1 replacement strain.

[0037] Figure 6 This refers to the clinical symptom score after mice were infected with the CpDHR1 replacement strain.

[0038] Figure 7 This represents the rate of weight loss in mice infected with the CpDHR1 replacement strain.

[0039] Figure 8 This is a virulence evaluation of the CpDHR1 replacement strain – mouse survival analysis. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0041] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0042] The pACT1::Cas9-GFP-U6::sgTK plasmid used in the examples was from the L. David Sibley laboratory at the University of Washington, and the cgd8_5420-Nluc-P2A-neo-mCh-cgd8_5420 plasmid was from our laboratory.

[0043] Typological identification of wild-type Cryptosporidium parvum subtype IIdA20G1 strains: 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 dairycalves. Transbound Emerg Dis. 2022;69:e1606-17. Example 1: Construction of Cryptosporidium microsporidium CpDHR1 replacement strain In the early stages of this invention, genetic hybridization was performed on two Cryptosporidium microsporidium isolates with different virulence. Using a single oocyst analysis system of their offspring, key genes associated with virulence differences were systematically identified, and gene substitution technology was used to verify the function of candidate genes in the infection and pathogenesis processes. Specifically, the CpDHR1 gene was identified as a potential virulence-related gene in both high polymorphism screening and positive selection analysis, and base differences were found in Cryptosporidium microsporidium strains with different virulence. The nucleotide sequence of the CpDHR1 gene in the highly virulent IIdA20G1 subtype Cryptosporidium strain is shown in SEQ ID NO: 1; the nucleotide sequence of the CpDHR1 gene in the low-virulence IIdA19G1 subtype strain is shown in SEQ ID NO: 2, with the gene number cgd5_4090. This gene is annotated to encode an SFII type helicase belonging to the "DHR1 / Ecm16p / Kurz HrpA family". Its homologous protein in humans and mice is DHX37, and its homologous protein in yeast is named DHR1. It possesses a conserved RNA helicase domain and participates in the ribosomal RNA assembly process. Therefore, this invention names this gene Cryptosporidium microsporidium DEAH / RHA RNA helicase DHR1.

[0044] 1. Insect strains The starting strain used in this embodiment is the wild-type, highly toxic subtype IIdA20G1 of *Cryptospora microsporum*, belonging to the genus *Cryptospora* of the family Cryptosporidium in the order Cryptosporidium. The construction strategy for the CpDHR1 gene replacement strain is as follows: Figure 1As shown, the CpDHR1 gene of the highly virulent IIdA20G1 subtype insect strain was replaced with the CpDHR1 gene of the low-virulent IIdA19G1 subtype insect strain.

[0045] 2. Construction of CRISPR replacement plasmid pAct::Cas9-U6::sgCpDHR1-1-U6::sgCpDHR1-2 (1) gRNA primer design CpDHR gRNA sequences were designed using the online tool at the gRNA design website (http: / / grna.ctegd.uga.edu / ): gRNA1 sequence is 5'-TCAAGTTGAATTTCTGATGA-3', PAM sequence is AGG; gRNA2 sequence is 5'-AATCTGATCAGAATAGTGGG-3', PAM sequence is AGG. Primers for constructing the double sgRNA plasmid were also designed and synthesized. Specific primer information is shown in Table 1 below.

[0046] Table 1. Primers required for constructing the double sgRNA plasmid pAct::Cas9-U6::sgGP601-U6::sgGP602

[0047] (2) Construction of single gRNA CRISPR plasmid Using pACT1::Cas9-GFP-U6::sgTK plasmid as a template, a single sgRNA CRISPR plasmid was constructed by linearizing the gRNA sequence by adding a homologous arm based on the principle of homologous recombination. The PCR reaction system and procedure for amplification are shown in Tables 2 and 3 below.

[0048] Table 2 PCR reaction system

[0049] Table 3 PCR reaction procedure

[0050] The PCR products were digested using the restriction endonuclease Dpn I from Novizan to remove the plasmid template. The reaction system and procedure are shown in Tables 4 and 5 below.

[0051] Table 4 Dpn I Reaction System

[0052] Table 5 Digestion reaction procedure

[0053] After the digestion was completed, the PCR products were recovered using the Novizan PCR Product Purification Kit. The concentration was then measured using a Thermo Fisher Scientific NanoDrop 2000 nucleic acid protein analyzer. The ligation was then performed using a TransGen multi-fragment ligation kit. The ligation system is shown in Table 6 below.

[0054] Table 6 Connection System

[0055] In the table, X represents the amount of amplified fragment used, specifically (0.02 × number of bases) ng / product recovery concentration. The reaction mixture was thoroughly mixed, briefly centrifuged at 37°C for 30 min, and then immediately placed on ice. It was then transformed into DH5α competent cells to construct plasmids. The CRISPR plasmid containing gRNA1 was named CpDHR CRISPR1, and the CRISPR plasmid containing gRNA2 was named CpDHR CRISPR2.

[0056] (3) Construction of dual gRNA CRISPR plasmids Using the CpDHR CRISPR1 plasmid as a template, PCR amplification of the plasmid backbone was performed using primers gRNA1-F / R (Table 1), with reaction conditions and procedures identical to those in Tables 2 and 3. Similarly, using the CpDHR CRISPR2 plasmid as a template, the gRNA fragment was amplified using primers gRNA2-F / R (Table 1), with the reaction system and procedure identical to the previous steps. The PCR products were then detected by 1.2% agarose gel electrophoresis. After gel extraction and recovery of the target band, the two correct fragments were ligated using a rapid ligation kit from TransGen Biotech, following the same construction method. The successfully constructed plasmid was named pAct::Cas9-U6::sgCpDHR1-1-U6::sgCpDHR1-2.

[0057] 3. Construction of homology repair plasmid CpDHR1-Nluc-P2A-neo-mCh-CpDHR1 (1) Extraction of Cryptosporidium microsporidium gDNA Genomic DNA was extracted from oocyst samples of Cryptosporidium microsporidium subtype IIdA20G1 strains according to the instructions of QIAGEN (Germany).

[0058] (2) PCR amplification of the target fragment Using the Cryptosporidium microsporidium oocyst DNA and template plasmid cgd8-5420-Nluc-P2A-neo-mCh-cgd8-5420 prepared above as templates (constructed using conventional methods in the field, with homologous arms at both ends and CpDHR (GD-IIdA19G1), luciferase assay gene Nluc, drug screening gene Neo, and red fluorescent protein mCherry linked in the middle), the corresponding target fragments were amplified using primers designed in Table 7 below. The specific amplification system and procedure are the same as those shown in Tables 2 and 3.

[0059] Table 7 Primers required for constructing the homologous template plasmid CpDHR1-Nluc-P2A-neo-mCh-CpDHR1

[0060] (3) Recovery of the target fragment The PCR products were then detected by 1.2% agarose gel electrophoresis, using the FastPure Gel DNA Extraction Mini Kit (DC301) from Nanjing Novizan Biotechnology Co., Ltd. 01) The target fragment was recovered according to the kit instructions, and the concentration of the recovered fragment was detected using a nucleic acid protein concentration analyzer (NanoDrop 2000).

[0061] (4) Construction of CpDHR1-Nluc-P2A-neo-mCh-CpDHR1 homologous template plasmid Following the instructions of the multi-fragment rapid cloning kit from All Gold Corporation (operated according to the manufacturer's instructions), the four fragments prepared were ligated using homologous recombination. The multi-fragment ligation reaction system is shown in Table 6. After mixing the reaction solution and briefly centrifuging, ligation was performed at 37°C for 30 minutes. The cells were immediately placed on ice after ligation. The ligation product was then transformed into competent cells, and the transformed bacterial culture was sent for sequencing verification. Positive clones with correct sequencing results were selected, plasmids were extracted, and stored at -20°C for later use. The plasmid CpDHR1-Nluc-P2A-neo-mCh-CpDHR1 was successfully constructed.

[0062] 4. Construction of Cryptosporidium microsporidium CpDHR1 gene replacement strain (1) Obtaining oocysts of Cryptosporidium microsporidium subtype IIdA20G1 strain Grinding the feces: Place an appropriate amount of mouse positive fecal samples soaked in 2.5% potassium dichromate solution and a magnetic stirrer together in a 250 mL wide-mouth glass container, and pour in an appropriate amount of potassium dichromate to cover the feces. Then place the container on a magnetic stirrer in a 4°C refrigerator and stir for 6-8 hours. The fecal sample should be in a homogenous suspension state.

[0063] Fecal sieving: The fecal suspension was passed through a 20-mesh coarse sieve and a 60-mesh fine sieve, stirring with a Pasteur tube during sieving. The sieves were then rinsed with pure water until the fecal residue became colorless. The volume was then adjusted to 1 L. After stirring thoroughly, the suspension was allowed to stand for 10 minutes. The supernatant was then slowly poured into a 500 mL centrifuge tube. The 500 mL centrifuge tube was balanced and centrifuged at 4200 rpm (rapid acceleration 8, falling speed 6) for 10 minutes. After centrifugation, the supernatant was discarded, and the precipitate was resuspended in 120 mL of pure water to prepare a crude oocyst suspension.

[0064] Sucrose density gradient centrifugation: First, prepare a 1:2 sucrose solution (80 mL saturated sucrose + 160 mL pure water) and a 1:4 sucrose solution (50 mL saturated sucrose + 200 mL pure water). Add Tween-80 to the saturated sucrose solution, with a Tween-80 to sucrose solution ratio of 1:100. Prepare 12 50 mL centrifuge tubes in advance. Add 20 mL of the 1:4 sucrose solution to each tube first, then slowly add 20 mL of the 1:2 sucrose solution from the bottom of the tube using a 15 mL Pasteur tube. At this point, a clear stratification interface will appear between the different concentrations of sucrose solution as the 1:2 sucrose solution is added. Then, gently add 10 mL of crude oocyst suspension to the upper interface of the 1:4 sucrose solution using a Pasteur tube. After balancing, centrifuge at 1000 × g, 4℃ for 25 min, with an acceleration rate of 1 and a deceleration rate of 1.

[0065] Initial collection of oocyst fluid: After centrifugation, discard the upper 15 mL of liquid. Then, starting from the 20 mL mark, aspirate the oocyst band until the liquid level in the centrifuge tube is below 7.5 mL. Transfer the aspirated oocyst fluid to a new 50 mL centrifuge tube, then add pure water to bring the volume to 50 mL, and invert the tube several times. After balancing, centrifuge at 3095 × g, 4℃ for 10 min, with an ascending speed of 8 and a descending speed of 6. After centrifugation, discard the supernatant in the 50 mL centrifuge tube, add a small amount of pure water (no more than 5 mL) to resuspend the precipitate to prepare an oocyst suspension, and collect it in a separate tube.

[0066] Cesium chloride density gradient centrifugation: Add 1 mL of pre-cooled cesium chloride solution to a 1.5 mL low-adsorption centrifuge tube, then gently add 500 μL of oocyst suspension to the surface of the cesium chloride solution, balance, and centrifuge at 13200 rpm, 4℃ for 3 min.

[0067] Aspirating the oocyst bands: After centrifugation, white oocyst bands can be clearly observed at the 1 mL mark in a 1.5 mL low-absorption centrifuge tube. Use a 200 μL pipette tip to repeatedly aspirate the oocyst bands, transferring the aspirated oocyst fluid to a new 1.5 mL centrifuge tube until the oocyst bands are no longer visible. Then, add pre-cooled ultrapure water to the 1.5 mL mark, repeatedly invert to mix, balance, and centrifuge at 13200 rpm, 4℃ for 3 min.

[0068] Oocyst collection: After centrifugation, discard the supernatant in the 1.5 mL centrifuge tube and resuspend the oocyst in pre-cooled ultrapure water. Collect the oocyst in another tube and add pre-cooled ultrapure water to the 1.5 mL mark. Balance the mixture and centrifuge at 13200 rpm, 4℃ for 3 min. After centrifugation, discard the supernatant, add 1 mL of pre-cooled PBS to resuspend the oocyst, and finally add 10 μL of penicillin-streptomycin-amphoteric B solution (100 ×).

[0069] Oocyte counting and preservation: 10 μL of the mixed ovarian fluid was added to a hemocytometer and counted under an optical microscope. After counting, the information was marked and stored at 4°C.

[0070] (2) Obtaining fresh sporozoites of Cryptosporidium microlucgiformes subtype IIdA20G1 strain Take 1.5 × 10 7 Fresh oocysts were placed on ice, and 200 μL of Clorox sterilizing solution and 600 μL of PBS were added. The mixture was incubated on ice for 10 minutes. Then, the mixture was centrifuged at 13200 rpm for 3 minutes at 4°C, and the supernatant was completely discarded. The oocyst precipitate was washed three times with PBS. Next, the oocysts were resuspended in 400 μL of 1% BSA, and then 400 μL of 1.5% taurine solution was added. The mixture was incubated in a 37°C water bath for 50 minutes to complete decapsulation. After decapsulation, the sample was centrifuged at 13200 rpm for 3 minutes, and the supernatant was discarded. The precipitate was resuspended in 1 mL of room temperature PBS to prepare a sporozoite suspension. This suspension was then centrifuged again under the same conditions, and the supernatant was discarded. This washing step was repeated twice to obtain sporozoites.

[0071] (3) Electroporation of sporozoites The SF Cell Line 4D-Nucleofector kit from Lonza, Germany, was used. TM X Kit L, catalog number V4XC-2024), resuspend the sporozoites in 80 μL of electroporation buffer (buffer composition: 65.6 μL SF buffer + 14.4 μL S1 buffer). See Table 8 for the specific reaction system configuration. Both the CRISPR knockout plasmid and the homology repair plasmid were used at 50 μg, and their concentrations were adjusted to 5000 ng / μL before electroporation.

[0072] Table 8 100 µL Electrotransfer System

[0073] The electroporation instrument was turned on in advance, and then the electroporation cup was placed in it. Electroporation was performed using the EH100 preset program of the Lonza Nucleofector system. After the electroporation was completed, 4 μL of electroporation solution was taken out and placed in a PCR tube to infect a 24-well cell plate filled with HCT-8 cells. The electroporation efficiency was evaluated 24 h after infection.

[0074] (4) Infecting mice One 4-6 week old IFN-γ KO mouse was administered 200 μL of 8% sodium bicarbonate via gavage to neutralize gastric acid. Five minutes later, the remaining sporozoite suspension was administered to the mouse via gavage. Then, 200 μL of PBS was used to resuspend the remaining sporozoites in the electroporation vessel, and the resuspended suspension was administered to the mouse via gavage. Eighteen hours after inoculation, the mice's drinking water was replaced with a 16 g / L paromomycin solution for drug screening.

[0075] (5) Detection of luciferase value Starting from day 5 after electroporation, fresh fecal samples were collected from mice every two days and placed in 1.5 mL centrifuge tubes. 1 mL of fecal lysis buffer and 10 fragmentation beads were added to the tubes, and the samples were thoroughly homogenized using a tissue homogenizer. The homogenized samples were centrifuged at 19000 × g for 1 minute to separate solid impurities from the supernatant. Simultaneously, 50 μL of luciferase substrate buffer and 1 μL of substrate were added to the reaction wells of a 96-well microplate. Then, 50 μL of the centrifuged supernatant was added to the corresponding well and gently mixed by pipetting. The reaction system was incubated in the dark for 3 minutes, and then immediately placed in a microplate reader to detect the chemiluminescent signal. A detection value greater than 300 was generally used as the threshold for a positive result in mouse oocyst excretion. Once the mouse fecal luciferase test was positive, fecal samples were continuously collected daily to enrich Cryptosporidium oocysts for subsequent purification and ultimately to obtain the CpDHR1 replacement strain.

[0076] (6) Identification of CpDHR1 replacement strains Three primer pairs (PCR1, PCR2, and PCR3) were designed and synthesized for molecular identification of the purified gene-edited parasite strain. The specific primer sequences are shown in Table 9 below. The reaction system and procedure used for PCR detection are the same as those in Tables 2 and 3.

[0077] Table 9 Primers for PCR identification of CpDHR1 replacement strains

[0078] The results of the identification are as follows Figure 2As shown, both PCR1 and PCR2 amplified the expected bands, indicating that the expression cassette containing the mCherry red fluorescent protein gene, the neomycin resistance gene Neo, and the luciferase reporter gene Nluc has been successfully integrated into the CpDHR1 target site of the Cryptosporidium microsporidium genome. PCR3 was used to detect the substitution of the CpDHR1 gene coding region (CDS), and the sequenced region was analyzed. The sequencing results are shown below. Figure 3 As shown, this indicates that the CpDHR1 gene replacement strain has been successfully constructed.

[0079] Example 2: In vitro growth experiment of Cryptosporidium microsporidium strain with CpDHR1 gene replacement In infected samples from the same gene-edited parasite strain, luciferase activity was linearly positively correlated with the number of parasites, increasing with the number of parasites. Since the proliferation cycle of Cryptosporidium after invading host cells is relatively fixed, the effect of gene editing on parasite proliferation can be indirectly assessed by comparing luciferase expression levels at different time points. If the deletion of the target gene inhibits parasite proliferation, a decrease in proliferation rate may be observed at specific developmental stages related to its function. Based on the above principles, this embodiment systematically evaluated the growth phenotype of the gene-edited parasite strain through in vitro culture experiments.

[0080] 1. In vitro infection HCT-8 cells (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number CBP60030) were infected using oocysts of the CpDHR1 gene-replaced strain obtained in Example 1. Cells were cultured in 1640 complete medium containing 2% fetal bovine serum. Cryptosporidium inoculation was performed when the cells reached confluence of 60% or more. Fresh oocysts were placed on ice, and 200 μL of Clorox sterile solution and 600 μL of PBS were added. The cells were incubated on ice for 10 minutes; centrifuged at 13200 rpm for 3 minutes at 4°C, and the supernatant was completely discarded; the oocyst pellet was washed three times with PBS; and the oocysts were resuspended in 1640 medium containing 2% FBS in a clean bench. After infecting the cells, the original HCT-8 cell culture medium was discarded, and 50,000 treated oocysts were inoculated into each well. The highly virulent CpDHR1-HA strain (carrying Nluc fluorescent and drug screening tags) was used as a control in this experiment.

[0081] 2. Luciferase assay Each experimental group had four replicates, and the luciferase values ​​of each well were measured at 3, 12, 24, and 48 h post-infection. The luciferase assay method was the same as in Experiment 1. After the assay was completed, the data were statistically analyzed using Graph Pad 10.0.

[0082] The measurement results are as follows Figure 4As shown, at 3 h and 12 h, there was no difference in growth between the CpDHR1-replaced strain and the CpDHR1-HA strain; however, at 24 h and 48 h, there was a significant difference: the insect load growth of the CpDHR1-replaced strain was significantly slower, and the luciferase detection values ​​were significantly different from those of the CpDHR1-HA strain. P The value <0.0001 indicates that the replacement of CpDHR1 can inhibit the growth of the parasite and reduce the proliferation capacity of Cryptosporidium.

[0083] Example 3: In vivo infection experiment of Cryptosporidium microsporidium CpDHR1 replacement strain In vivo infection experiments are a key method for assessing the pathogenicity of Cryptosporidium microsporidium. This study systematically evaluated the virulence phenotype of the strain by detecting in vivo parasite load, fecal oocyst excretion, and survival rate of infected mice.

[0084] 1. Test treatment GKO mice aged 3 to 5 weeks were selected for the experiment. Each mouse was housed individually, with 10 mice in each group, ensuring that the average weight of each group was approximately the same. A CpDHR1 replacement group was established: each mouse was inoculated with 1000 fresh oocysts of the CpDHR1 replacement strain; a control group was established: each mouse was inoculated with 1000 fresh oocysts of the CpDHR1-HA strain (same as in Example 2); and a blank group was established: no oocysts were inoculated. During the experiment, all mice were continuously treated with paromomycin (concentration 16 g / L) via drinking water.

[0085] 2. Determination of oocyst excretion intensity Oocyst excretion intensity was determined by fresh fecal luciferase values. Fresh feces were collected every two days starting from the second day after infection to determine the fresh fecal luciferase values. The luciferase determination method was the same as in Example 1, and the oocyst excretion intensity change curve was plotted using Graph Pad 10.0 software.

[0086] The results are as follows Figure 5 As shown, the excretion of oocysts by the CpDHR1 replacement strain increased slowly from 4 to 10 days after infection. Although there was no difference in the excretion volume between the two strains during the peak excretion period (12 to 18 days after infection), the overall excretion volume of oocysts by the CpDHR1 replacement strain was less than that of the control group.

[0087] 3. Monitoring of clinical symptoms in mice Clinical symptom scoring: The clinical symptoms of each mouse were scored every other day to determine the impact of CpDHR1 replacement on the pathogenicity of Cryptosporidium parvum. The scoring criteria were as follows: 1 point, the mouse was in good spirits and active; 2 points, the mouse was depressed and had a low activity rate; 3 points, the mouse was lethargic, arched its back, and would only run when touched; 4 points, the mouse had an arched back and could not move even when touched; 5 points, the mouse was in very poor spirits, unsteady on its feet, and almost motionless.

[0088] The scoring statistics are as follows Figure 6 As shown, a standardized scoring system was used to score the clinical symptoms of each mouse. Mice in the CpDHR1-HA group exhibited clinical symptoms such as lethargy, arched back, ruffled fur, and fecal adhesion with bleeding at DPI 10. Mice in the CpDHR1 replacement group only showed these clinical symptoms at DPI 12, and the symptoms were milder. This indicates that CpDHR1 replacement alleviates the clinical symptoms of Cryptosporidium parvum infection in mice, demonstrating a significant attenuation effect.

[0089] 4. Monitoring mouse body weight The body weight of mice was recorded daily during the experiment to determine the effect of CpDHR1 replacement on the body weight of Cryptosporidium microphyllum. Statistical results are as follows: Figure 7 As shown, the mice in the DPI 8 replacement group began to lose weight, indicating that replacing CpDHR1 would reduce the rate of weight loss in mice infected with Cryptosporidium microsporidium.

[0090] 5. Plotting mouse survival curves The mental status and survival of the mice were recorded daily during the experiment, and the time of death was precisely recorded. Based on the survival data, survival rates were plotted as survival curves using Graph Pad 10.0 software to assess survival differences between different infection groups. The results are as follows: Figure 8 As shown, mice in the CpDHR1-HA group began to die gradually from DPI 13, and all died by DPI 19; while mice in the CpDHR1 replacement group also began to die gradually from DPI 13, but all died by DPI 22. Therefore, compared with GKO mice infected with the CpDHR1-HA strain, mice infected with the CpDHR1 replacement strain had a longer survival time and a significant attenuation effect.

[0091] In summary, this invention successfully obtained a CpDHR1-substituted strain of Cryptosporidium parvum by replacing the CpDHR1 gene using CRISPR / Cas9 technology, and found that the CpDHR1 gene is related to the virulence of the parasite. The CpDHR1-substituted strain exhibited significantly reduced infectivity and virulence in IFN-γ knockout (GKO) mice. Under in vitro growth conditions, the CpDHR1-substituted strain showed decreased proliferation capacity and inhibited sporozoite growth. Under in vivo growth conditions, after infecting GKO mouse models with the CpDHR1-substituted strain, the parasite load decreased, the oocyst excretion rate decreased, clinical symptoms were alleviated, and survival time was prolonged, thus reducing the virulence of the sporozoites. This indicates that the substitution of CpDHR1 affects the virulence of Cryptosporidium parvum and can serve as a novel candidate strain for attenuated vaccine preparation.

[0092] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A Cryptosporidium microsporidium CpDHR1 gene-replaced strain, characterized in that, The CpDHR1 gene of a highly virulent Cryptosporidium strain was replaced with the CpDHR1 gene of a low-virulence Cryptosporidium strain using CRISPR / Cas9 gene editing technology, thereby obtaining a Cryptosporidium strain with a replaced CpDHR1 gene. The nucleotide sequence of the CpDHR1 gene of the highly virulent strain is shown in SEQ ID NO: 1; the nucleotide sequence of the CpDHR1 gene of the low-virulence strain is shown in SEQ ID NO:

2.

2. The method for preparing the CpDHR1 gene-replaced insect strain according to claim 1, characterized in that, Includes the following steps: S1. Using pAct::Cas9-GFP-U6::sgTK plasmid as template, and employing sgRNA primers of the low-virulence insect strain CpDHR1 gene, a double sgRNA CRISPR plasmid pAct::Cas9-U6::sg CpDHR1-1-U6::sg CpDHR1-2 was constructed. S2. Using the plasmid cgd8_5420-Nluc-P2A-neo-mCh-cgd8_5420 and the gDNA of the wild-type Cryptosporidium microsporum strain as templates, and using the homologous recombination template primers of the low-virulence strain CpDHR1 gene, the homologous recombination plasmid CpDHR1-Nluc-P2A-neo-mCh-CpDHR1 was constructed. S3. The pAct::Cas9-U6::sg CpDHR1-1-U6::sg CpDHR1-2 plasmid and the CpDHR1-Nluc-P2A-neo-mCh-CpDHR1 homologous recombinant plasmid were co-transfected into wild-type Cryptosporidium microsporum strains. The Cryptosporidium microsporum CpDHR1 gene replacement strains were obtained by drug screening and PCR identification.

3. The method according to claim 2, characterized in that, The primer sequences for the CpDHR1 gene sgRNA in step S1 are shown in SEQ ID NO: 3~10.

4. The method according to claim 2, characterized in that, The primer sequences for homologous recombination of the CpDHR1 gene in step S2 are shown in SEQ ID NO: 11~20.

5. The method according to claim 2, characterized in that, In step S3, the wild-type Cryptosporidium microsporidium strain is the Cryptosporidium microsporidium IIdA20G1 subtype strain.

6. The application of a formulation replacing the highly virulent CpDHR1 gene of Cryptosporidium parvum in the construction of Cryptosporidium parvum CpDHR1 gene replacement strains or in the preparation of Cryptosporidium parvum vaccines, characterized in that... The formulation replaces the CpDHR1 gene of a highly virulent insect strain with the CpDHR1 gene of a low-virulent insect strain; the nucleotide sequence of the CpDHR1 gene of the highly virulent insect strain is shown in SEQ ID NO: 1; the nucleotide sequence of the CpDHR1 gene of the low-virulent insect strain is shown in SEQ ID NO:

2.

7. The application of a formulation replacing the CpDHR1 gene of Cryptosporidium microsporidium in the preparation of products that inhibit the growth of Cryptosporidium microsporidium, characterized in that, The formulation replaces the CpDHR1 gene of a highly virulent insect strain with the CpDHR1 gene of a low-virulent insect strain; the nucleotide sequence of the CpDHR1 gene of the highly virulent insect strain is shown in SEQ ID NO: 1; the nucleotide sequence of the CpDHR1 gene of the low-virulent insect strain is shown in SEQ ID NO:

2.

8. The application of a knockout or replacement agent targeting the CpDHR1 gene of a highly virulent Cryptosporidium strain in the preparation of drugs for the prevention of Cryptosporidium infection, characterized in that... The nucleotide sequence of the highly virulent insect strain CpDHR1 gene is shown in SEQ ID NO:

1.

9. The use of the Cryptosporidium microsporidium CpDHR1 gene replacement strain according to claim 1 in the preparation of Cryptosporidium microsporidium attenuated products.

10. A Cryptosporidium microsporidium attenuated product, characterized in that, The strain containing the Cryptosporidium microsporidium CpDHR1 gene replacement as described in claim 1.

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