Cryptosporidium parvum CpCyP23 deleted strain as well as construction method and application thereof

Abstract

The invention is applicable to the technical field of gene engineering, and provides a cryptosporidium parvum CpCyP23 deleted strain as well as a construction method and application thereof, the construction method comprises the following steps: a CpCyP23 gene is knocked out by using a CRISPR / Cas9 technology to obtain the cryptosporidium parvum CpCyP23 deleted strain, and the sequence of the CpCyP23 gene is shown as SEQ ID NO: 27. The invention provides the construction method of the cryptosporidium parvum CpCyP23 deleted strain and the CpCyP23 deleted strain constructed by the construction method, and proves that the deletion of the cyclophilin CpCyP23 of the cryptosporidium parvum delays the ectogenesis of the cryptosporidium parvum, and the CpCyP23 deleted strain can reduce the pathogenicity of the cryptosporidium parvum. The result shows that the CpCyP23 is a vaccine target spot for intervening cryptosporidium infection, and the deletion strain of the CpCyP23 can be used as a cryptosporidium vaccine candidate strain.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and in particular relates to a Cryptosporidium parvum CpCyP23 deletion strain, a construction method and an application thereof. Background Art

[0002] Cryptosporidium is an important zoonotic parasite that poses a serious threat to human health and animal husbandry development. Cryptosporidium parvum , C. parvum Cryptosporidium is a major cause of severe diarrhea in humans and animals, and there is no effective preventive or treatment. Treatment options for cryptosporidiosis are limited, and the drug nitazoxanide has limited clinical efficacy in malnourished children and immunocompromised individuals. Currently, there is no commercially available Cryptosporidium vaccine for the prevention of cryptosporidiosis.

[0003] Peptidyl-prolyl cis / trans isomerases (PPIase) is a class of enzymes that can catalyze the cis / trans isomerization of peptide-prolyl, PPIase plays a role in various biological processes, such as protein folding, protein transport, cell signaling, cell growth, etc., according to the different inhibitors, the PPIase superfamily is divided into four non-homologous protein families, namely FK506 binding protein (FKBPs), cyclophilin (CyPs), small virus protein (Pars) and protein phosphatase 2A phosphatase activator (PTPA). At present, the PPIase of many clinically important protozoa has been studied, such as Giardia, Trypanosoma, Leishmania, Plasmodium and Toxoplasma, which reveals that PPIase plays an important role in the survival, development and pathogenicity of these organisms: among them, PfKBP-35 of Plasmodium plays a role in the interaction of parasite-nucleus; TgCyP20 in Toxoplasma is a secreted protein, which interacts with cysteine-cysteine chemotactic factor receptor 5 and triggers the production of IL-12; and TgCyp18 can induce the production of nitric oxide, which plays a key role in inhibiting parasite replication and triggering bradyzoite development. However, there are few reports on the biological function of Cryptosporidium parvum PPIase. Cryptosporidium parvum genome contains 9 genes encoding PPIase, including 7 cyclophilin (CyP) type and 2 FK506 binding protein (FKBP) type. In the existing research, the localization of Cryptosporidium parvum PPIase was predicted by bioinformatics analysis, and the results showed that CpFKBP-34 and CpFKBP-34 were located in the nucleolus, CpCyP34 was located in the nucleus, CpCyP19 was located in the sheared body, and CpCyP18, CpCyP21.1, CpCyP21.2 and CpCyP23 were located in the cytoplasm; and in the existing technology, CpCyP23 was found in the extracellular vesicle of Cryptosporidium parvum oocyst. However, the function of CpCyP23 in Cryptosporidium parvum is still unknown, and whether it can be used as a vaccine target is also unknown. Since there is currently a lack of vaccine or gene deletion strain that can be used to prevent and control Cryptosporidium, it is necessary to develop more new gene deletion strains or vaccines for preventing Cryptosporidium infection, and to provide technical support for the prevention and control of cryptosporidiosis. SUMMARY

[0004] The purpose of the embodiment of the present application is to provide a method for constructing a Cryptosporidium parvum CpCyP23 deletion strain, which aims to solve the problems raised in the above background art.

[0005] The embodiment of the application is achieved in the following manner: a micro cryptosporidium CpCyP23 deletion strain is constructed by using CRISPR / Cas9 technology to knock out a CpCyP23 gene, wherein the sequence of the CpCyP23 gene is shown as SEQ ID NO: 27.

[0006] Preferably, the method comprises the following steps: The TK sgRNA in the A2-Aldo_Cas9_Ribo-GUIDE plasmid is replaced with sgRNA matched with the 108-127 bp region of the CpCyP23 gene, and a Q5 site-directed mutagenesis kit is used to generate the plasmid A2-Aldo_Cas9_Ribo-GUIDE-CpCyP23-KO. The upstream and downstream homologous arms are amplified from the micro cryptosporidium genomic DNA, and the upstream and downstream homologous arms are respectively a 988 bp fragment before the promoter of the CpCyP23 gene and a 893 bp fragment after the terminator, and the upstream and downstream homologous arms are respectively inserted into the Nluc gene tag before and the NeoR gene after the plasmid cpLICX3HA-eno-nluc-neo by means of Gibson assembly to obtain a repair plasmid cpLICX3HA-eno-nluc-neo-CpCyP23-KO for homologous recombination. The plasmid A2-Aldo_Cas9_Ribo-GUIDE-CpCyP23-KO and the plasmid cpLICX3HA-eno-nluc-neo-CpCyP23-KO are co-transfected into a wild-type micro cryptosporidium strain, and a micro cryptosporidium CpCyP23 deletion strain is obtained through paromomycin drug screening and PCR identification.

[0007] Preferably, in the step of replacing the TK sgRNA in the A2-Aldo_Cas9_Ribo-GUIDE plasmid with sgRNA matched with the 108-127 bp region of the CpCyP23 gene, the point mutation primer sequence used is shown as SEQ ID NO: 1-2.

[0008] Preferably, in the step of amplifying the upstream and downstream homologous arms from the micro cryptosporidium genomic DNA, the upstream and downstream homologous arms are respectively a 988 bp fragment before the promoter of the CpCyP23 gene and a 893 bp fragment after the terminator, and the upstream and downstream homologous arm amplification primer sequence used is shown as SEQ ID NO: 3-6.

[0009] Preferably, in the step of co-transfecting plasmid A2-Aldo_C one as9_Ribo-GUIDE-CpCyP23-KO and plasmid cpLICX3HA-eno-nluc-neo-CpCyP23-KO into a wild type Cryptosporidium parvum strain, the wild type Cryptosporidium parvum strain is a strain identified as IIaA15G2R1 by GP60 genotyping.

[0010] Another object of the embodiments of the present application is to provide a Cryptosporidium parvum CpCyP23 deletion strain constructed by the above construction method.

[0011] Another object of the embodiments of the present application is to provide an application of a Cryptosporidium parvum CpCyP23 deletion strain in preparing a drug against cryptosporidiosis or a vaccine for preventing Cryptosporidium infection.

[0012] The construction method of the Cryptosporidium parvum CpCyP23 deletion strain and the CpCyP23 deletion strain constructed by the construction method provided by the embodiments of the present application, and it is confirmed that the deletion of Cryptosporidium parvum cyclophilin CpCyP23 delays the in vitro development of Cryptosporidium parvum, the CpCyP23 deletion strain can reduce the pathogenicity of Cryptosporidium parvum, indicating that CpCyP23 is a vaccine target for intervention of Cryptosporidium infection, and the CpCyP23 deletion strain can be used as a Cryptosporidium vaccine candidate strain. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 The construction schematic diagram and results of the CpCyP23 deletion mutant strain provided for the embodiments of the present application are shown in the following table; wherein A is a CyP23 knockout schematic diagram; B is a PCR identification result of the CpCyP23 deletion strain; Figure 2 The analysis results of the expression and cellular localization of CyP23 provided for the embodiments of the present application are shown in the following table; wherein A is a CpCyP23 epitope labeling schematic diagram; B is a PCR identification result of the CpCyP23-HA gene locus; C is a Western blot identification result of CpCyP23-HA expression; Figure 3 The results of the influence of the deletion of the CpCyP23 gene on the in vitro development of Cryptosporidium parvum provided for the embodiments of the present application are shown in the following table (** p<0.01, **** p<0.0001); Figure 4Results of the influence of the CpCyP23 deletion strain provided in Embodiment 6 of the present application on mice; wherein A is the change in the luciferase value of the feces of IFN-γ deficient mice after infection with CpCyP23-KO and CpCyP23-HA oocysts (**** p<0.0001); B is the survival curve of IFN-γ deficient mice after infection with CpCyP23-KO and CpCyP23-HA oocysts; C and D are the body weight changes of IFN-γ deficient mice within 30 days after infection with CpCyP23-KO and CpCyP23-HA oocysts; Figure 5 Results of the influence of the CpCyP23 deletion strain provided in Embodiment 7 of the present application on the histology of the small intestine of mice; wherein A is the hematoxylin and eosin staining image of the ileum of IFN-γ deficient mice infected with CpCyP23-KO and CpCyP23-HA parasites taken by microscope; B is the ratio of villus length to crypt depth of the ileum of IFN-γ deficient mice infected with CpCyP23-KO and CpCyP23-HA parasites. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solutions and advantages of the present application clearer, the present application will be further described in detail below in combination with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0015] The human ileocecal cancer cells (HCT-8) (ATCC CCL-244) used in the embodiments of the present application were purchased from the Shanghai Branch of the Chinese Academy of Sciences; the micro cryptosporidium strain (GP60 genotyping identified as: IIaA15G2R1) was preserved and passed in the laboratory; the IFN-γ deficient mice were purchased from Shanghai South Model Organism Technology Co., Ltd.

[0016] The specific implementation of the present application will be described in detail below in combination with specific embodiments.

[0017] Embodiment 1, construction of a gene knockout CRISPR / Cas9 plasmid: To achieve the deletion of the CpCyP23 gene, the TK sgRNA (single guide RNA) in the A2-Aldo_Cas9_Ribo-GUIDE plasmid was replaced with an sgRNA matching the 108-127 bp region of the CpCyP23 gene (the sequence of the CpCyP23 gene is shown as SEQ ID NO: 27), and a Q5 site-directed mutagenesis kit (NEB, MA, USA) was used to generate the plasmid A2-Aldo_Cas9_Ribo-GUIDE-CpCyP23-KO; wherein the point mutation primers are shown in Table 1; To construct the repair DNA for CpCyP23 gene deletion, the 988 bp fragment before the promoter of CpCyP23 gene (upstream homologous arm) and the 893 bp fragment after the terminator of CpCyP23 gene (downstream homologous arm) were amplified from the genomic DNA of Cryptosporidium parvum, and the upstream and downstream homologous arms were inserted into the plasmid cpLICX3HA-eno-nluc-neo before the Nluc gene tag and after the NeoR gene, respectively, by Gibson assembly, and finally the repair plasmid cpLICX3HA-eno-nluc-neo-CpCyP23-KO for homologous recombination was obtained. The primers for amplifying the upstream and downstream homologous arms are shown in Table 1: Table 1 Primers for constructing gene knockout plasmid

[0018] Example 2, Construction of CpCyP23 deletion mutant strain: The CpCyP23 deletion mutant strain was constructed using CRISPR / Cas9 technology. The plasmid A2-Aldo_Cas9_Ribo-GUIDE-CpCyP23-KO and the plasmid cpLICX3HA-eno-nluc-neo-CpCyP23-KO constructed in Example 1 were co-transfected into the wild-type Cryptosporidium parvum strain, and the baronmycin drug screening and PCR identification were performed to obtain the Cryptosporidium parvum CpCyP23 deletion strain. The mutant was obtained by replacing the CyP23 gene with the nanoluciferase (Nluc) driven by the enolase promoter (eno) and the neomycin resistance gene (NeoR) (as shown in Figure 1 Fig. A); Identification: Under the selection pressure of baronmycin, the CpCyP23 deletion strain can be successfully screened in the IFN-γ deletion mouse. The fecal samples of mice infected with the CpCyP23 deletion strain were collected and the genomic DNA was extracted. PCR amplification was performed using the primers shown in Table 2 to obtain the product of the expected size, while no same band was detected in the WT strain (as shown in Figure 1 Fig. B), confirming the accuracy of the genomic integration; in addition, the primer using the CpCyP23 open reading frame as a template failed to amplify the CpCyP23 band in the knockout strain, while the expected size fragment was detected in the WT strain (as shown in Figure 1 Fig. B); the above results show that CyP23 has been successfully knocked out in the genome of Cryptosporidium parvum: Table 2 Primers for identifying the CpCyP23 knockout strain of Cryptosporidium parvum

[0019] Example 3, Construction of CRISPR / Cas9 plasmid for epitope tagging: To provide the sgRNA plasmid for the CpCyP23 gene, the plasmid A2-Aldo_Cas9_Ribo-GUIDE-cgd1_870-HA was generated by replacing the TK sgRNA in the A2-Aldo_Cas9_Ribo-GUIDE plasmid with an sgRNA matching the 80-99 bp region before the stop codon of the CpCyP23 gene using the Q5 site-directed mutagenesis kit (NEB, MA, USA). The CpCyP23 sgRNA was designed by the eukaryotic pathogen CRISPR guide RNA / DNA designer tool (http: / / grna.ctegd.uga.edu), and the point mutation primers are shown in Table 3. Since the Cryptosporidium parvum lacks the non-homologous end joining (NHEJ) mechanism to repair DNA double-strand breaks, when performing gene editing, it is necessary to repair the DNA double-strand breaks induced by Cas9 by providing DNA for homologous recombination, specifically: a 900 bp fragment before the CpCyP23 gene terminator (upstream homologous arm, sgRNA sequence has been mutated) and a 940 bp fragment after the terminator (downstream homologous arm) were amplified from the Cryptosporidium parvum genomic DNA. The upstream and downstream homologous arms were inserted into the HA tag before and NeoR gene in the plasmid cpLICX3HA-eno-nluc-neo by Gibson assembly, respectively, to finally obtain the repair plasmid cpLICX3HA-eno-nluc-neo-CpCyP23-HA for homologous recombination. The upstream and downstream homologous arm amplification primers are shown in Table 3. Table 3 Primers for constructing epitope-tagged plasmids

[0020] Example 4, Epitope tagging of CpCyP23: To analyze the expression and cellular localization of CyP23 in Cryptosporidium parvum, 3 hemagglutinin epitopes (3xHA) were fused to the C-terminus of CpCyP23 using CRISPR / Cas9 technology, and a nanoluciferase (Nluc) driven by an enolase promoter and a neomycin resistance gene (NeoR) were constructed downstream of it (as shown in Figure 2 The PCR products of the expected sizes of 897, 709 and 894 bp were obtained (as shown in Figure 2 In addition, HCT-8 cells were infected with WT and epitope-tagged strains, respectively, and total proteins were collected 48 h later for Western blot analysis. The results showed that a band of about 26 kDa appeared in the lane of the cell sample of the epitope-tagged strain, while no corresponding band was found in the lane of the WT sample (as shown inFigure 2 The results show that the C-terminal of CpCyP23 successfully integrated 3xHA tag, and the CpCyP23-HA fusion protein was successfully expressed within 48 h after the parasites infected the cells: Table 4 Identification primers of epitope-tagged Cryptosporidium parvum CpCyP23 strain

[0021] Example 5, Effect of CpCyP23 deletion on in vitro development of parasites: To evaluate the effect of CpCyP23 deletion on the development of Cryptosporidium parvum, the growth rates of epitope-tagged strain and CpCyP2 deletion strain were compared by luciferase assay, and two kinds of transgenic oocysts were used to infect HCT-8 cells, respectively, and the luminescence values were detected at the designated time points, and the results are shown in Figure 3 It can be seen that the fluorescence values of the epitope-tagged strain group were significantly higher than those of the CpCyP23 deletion strain group at the asexual reproduction stage (12 h and 24 h) and the sexual reproduction stage (36 h and 48 h); it is confirmed that the deletion of CpCyP23 inhibits the development of in vitro parasites, and CpCyP23 is essential for the in vitro development of parasites.

[0022] Example 6, Effect of CpCyP23 deletion strain on mice: To study the biological function of CpCyP23 gene, 5000 CpCyP23-KO and CpCyP23-HA transgenic oocysts were used to gavage 3-5 week-old IFN-γ deficient mice, respectively, and each group had 6 mice; after infection, paromomycin was added to the drinking water; from the 3rd day after infection, the mouse feces were collected, and the body weight and death time of the mice were recorded daily to construct the survival curve; at the same time, the collected mouse feces were subjected to luciferase assay to evaluate the oocyst discharge of the mice. The results are shown in Figure 4 It can be seen that the luciferase activity in the fecal samples of mice infected with CpCyP23-KO oocysts was significantly lower (p<0.0001) than that of mice infected with CpCyP23-HA oocysts during 8-20 dpi (as shown in Figure 4 The data are from 3 technical replicates of each group of mice (mean ± SD), and the two-way ANOVA with Sidak's correction for multiple comparisons was used to determine the significance); the results of the mouse weight detection showed that the body weight of mice infected with CpCyP23-HA oocysts decreased sharply, and showed severe infection symptoms such as disordered fur, arching back and soft stool, while the body weight of mice infected with CpCyP23-KO oocysts decreased slowly and remained unchanged at the late stage of the experiment (as shown in Figure 4As shown in Figures C and D, the data are from three replicates of each group of mice (mean ± SD). The data of the CpCyP23-HA group were measured until day 23, because all mice in this group died of infection after this time point) without obvious clinical symptoms. In addition, mice infected with CpCyP23-HA oocysts died earlier from infection (12 dpi), while mice infected with CpCyP23-KO oocysts survived longer, and no mice died until the experiment lasted for 30 days (as shown in Figure 4). Figure 4 (shown in B).

[0023] Example 7: Effects of CpCyP23-deficient strains on mouse small intestine histology: Two groups of IFN-γ-deficient mice (three mice per group) were infected with 5000 CpCyP23-KO and CyP23-HA transgenic oocysts, respectively. On day 15 post-infection, mice were euthanized, and the small intestine was isolated and flushed with PBS. The distal small intestine was fixed in 10% neutral-buffered formalin (Sangon) for 24 h and then transferred to a centrifuge tube containing 70% ethanol until processing. The distal small intestine was embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Slides were imaged using an Olympus BX51 light microscope equipped with a DP70 camera. Villus length and crypt depth were measured using ImageJ software (a total of 30 measurements per strain) to quantify the extent of intestinal damage. The results are as follows Figure 5 As shown, a few parasites were observed on the villi of the small intestine of mice in the CpCyP23-KO group, while many parasites were observed on the surface of the villi of the small intestine of mice in the CpCyP23-HA group (e.g. Figure 5 As shown in Figure A, black arrows indicate parasites attached to the villi of the small intestine, scale bar = 20 μm); the length of the villi and the depth of the crypts were measured, and the ratio of the two was calculated. The results showed that the ratio of the CpCyP23-HA group was significantly reduced compared with the CpCyP23-KO group (as shown in Figure 5). Figure 5 As shown in Figure B, the villus length and crypt depth of the small intestine of each group of mice were measured 30 times, and the results are shown as mean ± SD and tested by Mann-Whitney test), indicating that the loss of CpCyP23 significantly reduced the damage of the parasite to the intestine.

[0024] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for constructing a Cryptosporidium parvum CpCyP23 deletion strain, characterized in that: The following steps are involved: The CpCyP23 gene was knocked out using CRISPR / Cas9 technology to obtain a Cryptosporidium parvum CpCyP23 deleted strain. The CpCyP23 gene sequence is shown in SEQ ID NO:

27.

2. The method for constructing a Cryptosporidium parvum CpCyP23-deficient strain according to claim 1, characterized in that: The specific steps include: The TK sgRNA in the A2-Aldo_Cas9_Ribo-GUIDE plasmid was replaced with an sgRNA matching the 108-127 bp region of the CpCyP23 gene, and the plasmid A2-Aldo_Cas9_Ribo-GUIDE-CpCyP23-KO was generated using the Q5 site-directed mutagenesis kit; The upstream and downstream homology arms were amplified from the genomic DNA of Cryptosporidium parvum. The upstream and downstream homology arms were 988 bp before the CpCyP23 gene promoter and 893 bp after the terminator. These arms were then inserted into the plasmid cpLICX3HA-eno-nluc-neo before the Nluc gene tag and after the NeoR gene, respectively, by Gibson assembly to obtain the repair plasmid cpLICX3HA-eno-nluc-neo-CpCyP23-KO for homologous recombination. Plasmid A2-Aldo_Cas9_Ribo-GUIDE-CpCyP23-KO and plasmid cpLICX3HA-eno-nluc-neo-CpCyP23-KO were co-transfected into the wild-type Cryptosporidium parvum strain, and the CpCyP23-deficient Cryptosporidium parvum strain was obtained by paromomycin drug screening and PCR identification.

3. The method for constructing a Cryptosporidium parvum CpCyP23-deficient strain according to claim 2, characterized in that: In the step of replacing the TK sgRNA in the A2-Aldo_Cas9_Ribo-GUIDE plasmid with an sgRNA matching the 108-127 bp region of the CpCyP23 gene, the point mutation primer sequence used is shown in SEQ ID NO: 1-2.

4. The method for constructing a Cryptosporidium parvum CpCyP23-deficient strain according to claim 2, characterized in that: In the step of amplifying the upstream and downstream homology arms from the genomic DNA of Cryptosporidium parvum, wherein the upstream and downstream homology arms are respectively the 988 bp fragment before the CpCyP23 gene promoter and the 893 bp fragment after the terminator, the sequences of the upstream and downstream homology arm amplification primers used are shown in SEQ ID NOs: 3-6.

5. The method for constructing a Cryptosporidium parvum CpCyP23-deficient strain according to claim 2, characterized in that: In the step of co-transfecting the plasmid A2-Aldo_Cas9_Ribo-GUIDE-CpCyP23-KO and the plasmid cpLICX3HA-eno-nluc-neo-CpCyP23-KO into the wild-type Cryptosporidium parvum strain, the wild-type Cryptosporidium parvum strain is a strain identified as IIaA15G2R1 by GP60 typing.

6. A Cryptosporidium parvum CpCyP23 deletion strain, characterized in that: It is constructed by the construction method according to any one of claims 1 to 5.

7. Use of the Cryptosporidium parvum CpCyP23-deficient strain according to claim 6 in the preparation of a drug for preventing Cryptosporidium parvum infection or a vaccine for preventing Cryptosporidium parvum infection.

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