Recombinant coccidiosis vector for expressing VP2 protein and fluorescent label and detection method of recombinant coccidiosis vector
By inserting the VP2 gene and fluorescent protein gene into Eimeria tenella through CRISPR-Cas9 editing, a recombinant coccidia vector was constructed, which solved the problem that existing vaccines could not simultaneously immunize against infectious bursal disease and coccidiosis in chickens, and achieved stable expression of VP2 protein and enhanced immune effect.
Patent Information
- Application Number
- CN202311719607.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-14
- Publication Date
- 2026-04-14
AI Technical Summary
Current technologies are insufficient to effectively immunize chickens against both infectious bursal disease and Eimeria tenella simultaneously. Traditional vaccines face issues of drug resistance and drug residues, and there is a lack of vaccines that can immunize against both diseases at the same time.
Using the CRISPR-Cas9 gene editing system, the VP2 gene and the mCherry red fluorescent protein gene were inserted into the ETH_00009555 gene of Eimeria tenella to construct a recombinant coccidia vector. Stable expression and detection of the VP2 protein were achieved through fluorescence screening and PCR detection.
The constructed recombinant coccidia vector can stably and efficiently express VP2 protein, significantly enhance the body's humoral and cellular immune responses, and provide an effective means of simultaneously immunizing against infectious bursal disease and Eimeria tenella in chickens, reducing the workload of screening and improving editing efficiency.
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Figure CN121852469A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of gene editing technology, and in particular relates to a recombinant coccidia vector expressing VP2 protein and fluorescent tag and its detection method. Background Technology
[0002] The CRISPR-Cas9 system is a gene editing technology based on the adaptive immune mechanism of prokaryotes. It consists of two modules: a gRNA and its backbone that recognize specific DNA sequences, and the Cas9 protein that binds to and edits DNA. The CRISPR-Cas9 gene editing system is currently the most widely used gene editing system. Its principle is that the gRNA transcribed by CRISPR mediates the Cas nuclease targeting the target sequence and cutting it. CRISPR-Cas9 uses a small guide RNA to recognize the target sequence and guide the Cas9 protein to cut the target site, causing a double-strand break in the DNA. To prevent degradation, the broken DNA automatically initiates endogenous repair mechanisms. There are generally two types of endogenous repair mechanisms. In the non-homologous end joining repair mechanism, bases are often randomly inserted or deleted at the break point. If the mutation site is located in the protein coding region, incorrect mRNA will be transcribed, leading to translation failure or protein inactivation, thus achieving gene knockout. In the homologous recombination repair mechanism, under the condition of a repair template, targeted insertion, deletion, or mutation of a single base or long fragment can be achieved, resulting in gene recombination and knockout.
[0003] Infectious bursal disease (IBD) in chickens is a contagious disease caused by the infectious bursal disease virus (IBD virus). It primarily affects chicks. The virus infects the B lymphocytes of the bursa of Fabricius, causing immunosuppression and weakening the immune system. Infected chickens are highly susceptible to co-infection with other diseases, and their immune response to vaccines is reduced, preventing the body from producing antibodies and ultimately leading to immunization failure. The disease is acute, highly contagious, and spreads rapidly, making it difficult to control. Once an outbreak occurs, the entire flock is affected. It is not seasonal and can occur in all seasons, exhibiting endemic characteristics and spreading through the air to neighboring chicken houses. The disease damages the chicken's immune organs, leading to immunosuppression. Infected chickens often develop secondary diseases and symptoms other than IBD. Even with multiple vaccinations, their resistance to other bacterial or viral diseases remains reduced. In humid and hot environments, the disease can also cross-infect with parasitic diseases, severely hindering its control.
[0004] Coccidiosis is a common and highly damaging parasite found in chicken farms, causing coccidiosis that has a significant impact on the poultry industry. Coccidiosis is caused by one or more coccidia parasites infesting the chicken's body. Through continuous multiplication and destruction of intestinal epithelial cells, it leads to digestive disorders, anorexia, and ultimately weakens the chicken's immune system. *Eimeria tenella* is one of the most virulent coccidia, parasitizing the cecum and primarily affecting chicks under three months old. After chickens ingest coccidia oocysts, the coccidia implant in the intestines, multiply, and damage intestinal tissue. Coccidiosis in chickens presents with a sudden onset of symptoms, easily leading to large-scale infections and causing substantial losses to poultry farms. Due to its severe drug resistance and high recurrence rate, coccidiosis is difficult to treat; therefore, prevention and control are crucial.
[0005] Infectious bursal disease (IBD) and coccidiosis in chickens pose significant challenges to large-scale chicken farming. Traditional drug treatments, relied upon in the past, have led to drug resistance and residues with prolonged use. Current surveys show that coccidia have developed resistance to most anti-parasitic drugs. Simultaneously, the frequent emergence of new variants of the IBD virus has resulted in new outbreaks, and traditional vaccines are no longer effective against multiple variants. Therefore, developing vaccines against coccidiosis and IBD is crucial for their prevention and control.
[0006] Currently, novel vaccines developed for infectious bursal disease (IBD) in chickens mainly include recombinant subunit vaccines, live vector vaccines, and DNA vaccines. Vaccines for coccidiosis are relatively limited in type, and each vaccine only targets one type of coccidiosis. Achieving immunity against multiple coccidiosis diseases requires a considerable amount of time to establish an immune barrier. In large-scale poultry farms and hatcheries, rapid immunization is crucial for reducing the risk of disease in flocks. Currently, there is no vaccine that can simultaneously immunize against both IBD and Eimeria tenella. Summary of the Invention
[0007] To address the shortcomings of existing technologies, the present invention aims to provide a recombinant coccidia vector expressing VP2 protein and a fluorescent tag, along with its detection method. Using the CRISPR-Cas9 gene editing system, the VP2 gene and the mCherry red fluorescent protein gene can be inserted into the ETH_00009555 gene. The constructed recombinant coccidia can stably and efficiently express fluorescent signals and the VP2 protein. This invention successfully constructs a coccidia vector expressing VP2 protein and a fluorescent protein tag. This coccidia vector is easy to screen, exhibits autofluorescence under a fluorescence microscope, and is positive under PCR detection targeting the VP2 gene and the fluorescent protein gene.
[0008] To achieve this objective, the present invention adopts the following technical solution:
[0009] In a first aspect, the present invention provides an ETH_00009555 gene editing system, the ETH_00009555 gene editing system comprising a gene editing plasmid and a homologous recombination fragment;
[0010] The gene editing plasmid includes sgRNA and Cas9 targeting the ETH_00009555 gene;
[0011] The homologous recombination fragment includes the VP2 gene and a selection marker gene.
[0012] In this invention, the nucleotide sequence of the ETH_00009555 gene is obtained from NCBI.
[0013] Preferably, the sgRNA targeting the ETH_00009555 gene includes the sequence shown in SEQ ID No. 2.
[0014] Preferably, the nucleotide sequence of the VP2 gene includes the sequence shown in SEQ ID No. 1.
[0015] In this invention, the VP2 gene encodes the VP2 protein of a novel variant of infectious bursal disease virus (IBDV). The VP2 protein is a structural protein of IBDV, determining viral virulence and antigenic variation. It can induce apoptosis and generate neutralizing antibodies against the virus in poultry, thus protecting susceptible flocks from IBDV infection. It exhibits good immunogenicity. Inoculation with the VP2 protein can induce an immune response and enhance the resistance of chickens to IBDV.
[0016] Preferably, the screening marker gene is the mCherry red fluorescent protein gene.
[0017] The fluorescent selection marker gene in this invention is the mCherry red fluorescent protein gene. The mCherry red fluorescent protein emits a high fluorescence intensity, which is beneficial for screening.
[0018] In a second aspect, the present invention provides a recombinant coccidia vector containing the ETH_00009555 gene editing system described in the first aspect.
[0019] Preferably, the recombinant coccidia vector is a coccidia vector that has been edited using the ETH_00009555 gene editing system described in the first aspect, and has integrated the VP2 gene and the mCherry red fluorescent protein gene into the ETH_00009555 gene.
[0020] Thirdly, the present invention provides a method for detecting the recombinant coccidia vector described in the second aspect, the detection method comprising PCR amplification detection and / or fluorescence detection.
[0021] Preferably, the target genes detected by PCR amplification include the VP2 gene and / or the mCherry red fluorescent protein gene sequence.
[0022] Preferably, the fluorescence detection includes detection based on the fluorescence of the recombinant coccidia vector.
[0023] The present invention also provides a method for constructing the recombinant coccidia vector according to the second aspect, the method comprising:
[0024] Construct a gene-editing plasmid; construct a homologous recombination fragment; introduce the gene-editing plasmid and the homologous recombination fragment into coccidia sporozoites to infect animals; screen for positive clones to obtain the coccidia vector.
[0025] In this invention, the construction method is simple, highly operable, and has high editing efficiency, and can obtain insect strains with stable trait inheritance.
[0026] Preferably, the method for constructing the gene-editing plasmid includes:
[0027] The coding sequences of the sgRNA and Cas9 targeting the ETH_00009555 gene were amplified by PCR, and the coding sequences were ligated to obtain a gene editing plasmid containing the sgRNA and Cas9 targeting the ETH_00009555 gene.
[0028] Preferably, the method for constructing the homologous recombination fragment includes:
[0029] The VP2 gene and the selection marker gene were sequentially ligated to obtain a whole fragment. The whole fragment was then constructed into a cloning vector to obtain the homologous recombination plasmid. Using the obtained homologous recombination plasmid as a template, the homologous recombination fragment was amplified by PCR.
[0030] Preferably, the importation includes electrotransfection.
[0031] Preferably, the coccidia includes Eimeria tenella.
[0032] Preferably, the animal includes a chicken.
[0033] Preferably, the step of screening positive clones includes: screening based on fluorescent tag genes on homologous recombination fragments to obtain positive coccidia.
[0034] Preferably, the step of screening positive clones further includes orally inoculating the obtained positive coccidia and repeating the process for 4 to 5 generations to obtain a recombinant coccidia vector that stably expresses the VP2 protein.
[0035] In this invention, the mCherry red fluorescent protein gene is used as a fluorescent tag gene. During the screening process, positive coccidia (three or more sporozoites showing fluorescence) can be screened by observing fluorescence under a fluorescence microscope. The positive coccidia are then orally inoculated first with multiple oocysts and then with single oocysts. After repeating this process for four generations, a recombinant coccidia vector that naturally and stably expresses the VP2 protein is obtained.
[0036] As a preferred technical solution, the method for constructing the recombinant coccidia vector of the present invention includes the following steps:
[0037] (1) Constructing gene editing plasmids:
[0038] The coding sequences of the sgRNA and Cas9 targeting the ETH_00009555 gene were amplified by PCR, and the coding sequences were ligated to obtain the gene editing plasmid containing the sgRNA and Cas9 targeting the ETH_00009555 gene.
[0039] (2) Constructing homologous recombination fragments:
[0040] The VP2 gene and the fluorescent tag gene were sequentially linked to obtain a whole fragment, which was then constructed into a cloning vector to obtain the homologous recombination plasmid.
[0041] Using the obtained homologous recombination plasmid as a template, homologous recombination fragments were amplified by PCR;
[0042] (3) The gene editing plasmid containing the sgRNA and Cas9 targeting the ETH_00009555 gene and the homologous recombination fragment were introduced into the sporozoites of Eimeria tenella by electroporation, and chickens were infected with the sporozoites.
[0043] (4) Screening for positive clones:
[0044] Fluorescent signals were screened based on fluorescent marker genes on homologous recombination fragments to obtain positive fluorescent coccidia; the obtained positive fluorescent coccidia were orally inoculated, and after 4 generations, a recombinant coccidia vector stably expressing VP2 protein was obtained.
[0045] The order of steps (1) and (2) is not restricted by numbering.
[0046] Fourthly, the present invention provides a method for detecting the recombinant coccidia vector described in the second aspect, the detection method comprising PCR amplification detection and / or fluorescence detection.
[0047] Preferably, the target gene for PCR amplification detection includes the VP2 gene and / or fluorescent tag gene sequence.
[0048] Preferably, the fluorescence detection includes detection based on the fluorescence of the recombinant coccidia vector.
[0049] The detection method for the recombinant coccidia vector in this invention includes: extracting the genomic DNA of the recombinant coccidia vector, performing PCR detection, sequencing, and successfully recombinating both the VP2 gene and the fluorescent protein gene, proving that a recombinant coccidia vector expressing VP2 protein and fluorescent tag protein has been successfully constructed.
[0050] Fifthly, the present invention provides the use of the recombinant coccidia vector described in the second aspect in the preparation of a vaccine against infectious bursal disease in chickens.
[0051] In a sixth aspect, the present invention provides a coccidia vaccine comprising the recombinant coccidia vector and VP2 subunit vaccine described in the second aspect; the VP2 subunit vaccine comprising the IBDV mutant VP2 protein.
[0052] Preferably, the VP2 subunit vaccine further includes a white oil adjuvant.
[0053] In this invention, the coccidia vaccine comprises a recombinant coccidia vector and a VP2 subunit vaccine. In specific applications, animals are first immunized with the recombinant coccidia vector, followed by immunization with the VP2 subunit vaccine. This immunization method enhances the body's cellular immune response. When the recombinant coccidia vector and subunit vaccine are used for combined immunization, both humoral and cellular immune responses are significantly stronger than those immunized with the subunit vaccine alone, indicating that the recombinant coccidia vector provided by this invention has an immune-enhancing effect. Specifically, when immunizing animals with the recombinant coccidia vector, the coccidia vector is inoculated into chickens, propagated, purified, and sporulated to obtain fresh coccidia oocysts for immunization. The sporulated oocysts are diluted with water to a certain dose and administered orally. When immunizing animals with the VP2 subunit vaccine, it is administered subcutaneously.
[0054] It should be noted that the scientific and technical terms and their abbreviations used in this invention have the meanings commonly understood by those skilled in the art.
[0055] Compared with the prior art, the present invention has the following beneficial effects:
[0056] This invention avoids non-specific gene editing by specifically inserting the VP2 gene into the ETH_00009555 gene of *Eimeria tenella*, achieving high editing efficiency and low off-target rate. The constructed recombinant coccidia can pass the mutated gene to progeny coccidia through cell division, achieving stability and heritability of the gene mutation, reducing the workload of screening. This method is technically mature, simple to operate, has a high success rate, and good reproducibility. A spacer sequence is inserted between the VP2 gene and the selection marker gene to avoid the formation of a fusion protein between the VP2 protein and the selection marker protein, which is beneficial for the immunological activity of the VP2 protein. The fluorescence intensity emitted by mCherry red fluorescent protein is beneficial for the screening of positive individuals. When the recombinant coccidia vector and subunit vaccine are used for combined immunization, the humoral and cellular immune responses are significantly stronger than those of subunit vaccine alone, indicating a relatively stronger immune function; that is, coccidia can promote humoral and cellular immune responses. Attached Figure Description
[0057] Figure 1 This is the plasmid map of homologous recombination in Example 2.
[0058] Figure 2 The image shows a fluorescence micrograph of the coccidia vector in Example 2 (magnification = 200x).
[0059] Figure 3 The images shown are from the PCR detection of the coccidia vector in Example 3 (lane M: DL10000 DNA molecular weight standard; lane 1: coccidia vector DNA; lane 2: homologous recombination fragment; lane 3: maternal coccidia DNA; lane 4: negative control).
[0060] Figure 4 The results are the serum antibody level detection results from Example 4.
[0061] Figure 5 CD4 in Example 4 + T lymphocytes, CD8 + T lymphocyte test results.
[0062] Figure 6 CD4 in Example 4 + / CD8 + ratio. Detailed Implementation
[0063] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.
[0064] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.
[0065] The materials used in this embodiment were sourced as follows: PCR amplification reagents were purchased from Nanjing Novizan Biotechnology Co., Ltd.; plasmid extraction kits were purchased from Thermo Fisher Scientific; KLD reaction kits were purchased from NEB (Beijing) Co., Ltd.; electroporation buffers were purchased from Harvard Biosciences Co., Ltd.; Eimeria tenella were obtained from Foshan Zhengdian Biotechnology Co., Ltd.; and chicks were obtained from the SPF Laboratory Animal Center behind the headquarters of Wens Foodstuff Group in Xinxing County, Guangdong Province.
[0066] Example 1
[0067] This embodiment provides an ETH_00009555 gene editing system, which includes a gene editing plasmid and a homologous recombination fragment; the gene editing plasmid includes sgRNA and Cas9 targeting the ETH_00009555 gene; the homologous recombination fragment includes a 5' homologous arm, a 3' homologous arm, an SAG13 promoter, the VP2 gene of a novel variant of chicken infectious bursal disease virus, and the mCherry red fluorescent protein gene.
[0068] The nucleotide sequence of the novel variant of infectious bursal disease virus VP2 gene is shown in SEQ ID No. 1.
[0069] The nucleotide sequence of the sgRNA targeting the ETH_00009555 gene is shown in SEQ ID No. 2.
[0070] SEQ ID No. 2: gaataggccgaccagacggtg.
[0071] In this invention, an sgRNA sequence was designed targeting the exon 1 sequence of the Eimeria tenella gene sequence based on the ETH_00009555 gene sequence.
[0072] Example 2
[0073] This embodiment provides a recombinant coccidia. The recombinant coccidia vector is *Eimeria tenella*, which has been edited using the ETH_00009555 gene editing system described in Example 1, and into which the VP2 gene of a novel variant of infectious bursal disease virus (IBDV) has been inserted at a specific site. The recombinant coccidia vector is constructed using the following method:
[0074] (1) Construct gene editing plasmids containing sgRNA and Cas9 targeting the ETH_00009555 gene:
[0075] PCR amplification: The nucleotide sequence of the forward primer is shown in SEQ ID No. 3, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 4, the template is pSAG1-CAS9-U6 plasmid, and the PCR product is obtained.
[0076] SEQ ID No. 3: aataggccgaccagacggtggttttagagctagaaatagcaagttaa.
[0077] SEQ ID No. 4: aacttgacatccccatttacc.
[0078] The reaction system is shown in Table 1.
[0079] Table 1
[0080] reagents Dosage (μL) A mixture of enzymes, dNTPs, and buffer. 12.5 forward primer 1.25 reverse primer 1.25 The template is pSAG1-CAS9-U6 plasmid 1 water Supplement to 25
[0081] PCR reaction conditions are shown in Table 2:
[0082] Table 2
[0083]
[0084] The amplified fragment was 9600 bp in size and was detected by 1% agarose gel electrophoresis.
[0085] The PCR products were subjected to the KLD reaction and incubated at room temperature for 5 minutes. The reaction system for the KLD reaction is shown in Table 3.
[0086] Table 3
[0087] reagents Dosage (μL) reaction buffer 5 enzyme mixture 1 PCR products 1 water Supplement to 10
[0088] The obtained KLD reaction product was transformed into DH5α (100 μL) competent cells, plated on LB / Amp plates, and incubated upside down at 37°C for 12 hours.
[0089] Pick a single colony and place it in 5 mL of LB / Amp liquid medium. Incubate at 37°C and 180 rpm for 10 hours until the bacterial culture becomes turbid.
[0090] Mix 500 μL of bacterial culture with 500 μL of 40% glycerol and store the mixture at -20℃. Use the remaining bacterial culture for plasmid extraction and perform enzyme digestion identification on the extracted plasmid. The reaction system is as follows: 2.0 μL buffer, 1.0 μL SalI enzyme, 1.0 μL plasmid, and 16.0 μL sterile water. After reacting at 37℃ for 3 hours, perform agarose gel electrophoresis detection. The enzyme digestion product showing two bands (6kb and 3.7kb in size) indicates a positive clone.
[0091] The positive clones were sequenced and analyzed using universal primer M13 for one-way sequencing and M13 reverse primer for sequencing. The nucleotide sequence of the sequencing primer is shown in SEQ ID No. 4. The sequencing results showed that the target sequence had been completely replaced and the gene editing plasmid was successfully constructed.
[0092] (2) Constructing homologous recombination fragments:
[0093] The 5' homologous arm of the ETH_00009555 gene, the SAG13 promoter, the VP2 gene of a novel variant of chicken infectious bursal disease virus, the mCherry red fluorescent protein gene, the SAG13 terminator, and the 3' homologous arm of the ETH 00009555 gene were sequentially ligated to obtain a whole fragment. This whole fragment was then constructed into a vector to obtain the homologous recombination plasmid. The homologous recombination plasmid map is shown below. Figure 1 As shown, the nucleotide sequence of the homologous recombinant plasmid is shown in SEQ ID No. 5. The gene sequence was sent to BGI Genomics Co., Ltd. in Shenzhen for plasmid synthesis.
[0094] The homologous recombination plasmid was transformed into competent DH5α cells, and the plasmid was extracted. The homologous recombination plasmid was amplified by PCR using linearized primers. The nucleotide sequence of the forward primer of the linearized primer is shown in SEQ ID No. 6, and the nucleotide sequence of the reverse primer of the linearized primer is shown in SEQ ID No. 7, to obtain the homologous recombination fragment.
[0095] SEQ ID No. 6: tagtaggtatgggaggagtgtagttgg.
[0096] SEQ ID No. 7: tggatgcgtaggagtaataacacg.
[0097] (3) Transfection of Eimeria tenella spores and screening of positive clones:
[0098] Take 800,000 fresh and viable Eimeria coccidia spore suspensions and add them to an electroporation cuvette. Resuspend the worms in Cytomix (cell fusion buffer).
[0099] Cytomix (cell fusion buffer) consists of: 120 mmol / L KCl, 0.15 mmol / L CaCl2, 10 mmol / L K2HPO4 (pH 7.6), 25 mmol / L HEPES (pH 7.6), 2 mmol / L EGTA and 5 mmol / L MgCl2.
[0100] Gene editing plasmid and homologous recombination fragment were added to the suspended worm body, with the copy number ratio of the gene editing plasmid to the homologous recombination fragment being 1:1 and the plasmid concentration being approximately 300–400 ng / μL. The volume was adjusted to 800 μL with electroporation buffer and thoroughly mixed.
[0101] After placing the electroporation cup in an ice bath for 20 minutes and drying the cup walls, place it in a Bio-Rad electroporator. Set the voltage to 2000V, capacitance to 25μF, and capacitance to ∞, and perform 4-5 shocks. After each shock, remove the electroporation cup and mix it thoroughly by blowing. After electroporation, aspirate the liquid from the electroporation cup and rinse it with an appropriate amount of DMEM. Let the liquid in the electroporation cup and the DMEM rinsing solution stand for 15 minutes, then aspirate the entire solution into a 1mL syringe. Remove the syringe needle, dip it in glycerin, and infect 4-day-old SPF chickens (chickens raised in a barrier system or isolator that are free from major internationally or domestically prevalent chicken infectious disease pathogens) through the cloaca.
[0102] Chicken feces were collected on day 6 post-inoculation and purified to obtain oocysts. After oocyst sporulation, fluorescent oocysts were observed using an inverted fluorescence microscope to confirm fluorescent protein expression. Fluorescent single oocysts were screened and inoculated into chicks for further passage. After four generations, oocysts with a positive rate of over 95% were obtained, yielding stably genetically fluorescent oocysts, thus providing a stable genetically inherited coccidia vector. Coccidia that fluoresce under green light under a fluorescence microscope are considered coccidia vectors. Figure 2 As shown.
[0103] Example 3
[0104] This embodiment provides a method for detecting recombinant coccidia vectors. The detection method is used for Eimeria tenella, which was implanted with the VP2 gene of a novel variant of infectious bursal disease virus in chickens in Example 2.
[0105] The recombinant coccidia vector was detected using the following method:
[0106] The nucleotide sequence of the forward primer is shown in SEQ ID No. 6, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 7. The templates are coccidia vector DNA, coccidia vector cDNA, homologous recombinant plasmid, and parental DNA. A PCR product with a band size of 4562 bp was obtained. The reaction system is shown in Table 4. Figure 3 This is the PCR result.
[0107] Table 4
[0108] reagents Dosage (μL) A mixture of enzymes, dNTPs, and buffer. 12.5 forward primer 1.25 reverse primer 1.25 Template is DNA 1 water Supplement to 25
[0109] PCR reaction conditions are shown in Table 5:
[0110] Table 5
[0111]
[0112] The amplified fragment was 4562 bp in size and was detected by 1% agarose gel electrophoresis.
[0113] like Figure 3 As shown, the results indicate that the VP2 gene was successfully recombined in Eimeria tenella, and a recombinant coccidia vector was obtained.
[0114] Example 4
[0115] This embodiment provides a method for enhancing the immune effect of a recombinant coccidia vector on a VP2 subunit vaccine (prepared by expressing the VP2 gene (SEQ ID No. 1) of a novel IBDV variant strain in prokaryotes in Escherichia coli and then emulsifying it with white oil adjuvant).
[0116] (1) Experimental grouping and immunization procedure: Chickens were inoculated with EtVP2, a novel variant of the infectious bursal disease virus (IBDV) with the gene of VP2, and the parent strain (EtMZ). The strains were then propagated, purified, and sporulated to obtain fresh coccidia oocysts for animal experiments.
[0117] Eighty-four 3-day-old SPF chickens were randomly divided into 6 groups, and each chicken was numbered and weighed.
[0118] Groups 1 and 2 were given 500 EtVP2 sporulated oocysts by gavage at 3 days old; Groups 3 and 4 were given 500 EtMZ sporulated oocysts by gavage at 3 days old.
[0119] At 10 days of age, groups 1 and 2 received 5000 EtVP2 sporulated oocysts via gavage, while groups 3 and 4 received 5000 EtMZ sporulated oocysts via gavage. Additionally, groups 2, 4, and 5 received 0.1 mL of IBDV VP2 recombinant subunit vaccine (rVP2) subcutaneously. Group 6 received no treatment. At 17 days of age, groups 1 to 4 received 10,000 corresponding sporulated oocysts via gavage, while other groups received no treatment. The experimental groupings and immunization schedule are shown in Table 6.
[0120] Table 6
[0121] Group number Group Name Quantity (pieces) One exemption Two free Three exemptions 1 EtVP2 12 <![CDATA[500 a ]]> <![CDATA[5000 a ]]> <![CDATA[10000 a ]]> 2 EtVP2+rVP2 12 <![CDATA[500 a ]]> <![CDATA[5000 a +0.1 c ]]> <![CDATA[10000 a ]]> 3 EtMZ 12 <![CDATA[500 b ]]> <![CDATA[5000 b ]]> <![CDATA[10000 b ]]> 4 EtMZ+rVP2 12 <![CDATA[500 b ]]> <![CDATA[5000 b +0.1 c ]]> <![CDATA[10000 b ]]> 5 rVP2 12 — <![CDATA[0.1 c ]]> — 6 Blank group 12 — — —
[0122] Note: a, EtVP2 vector immunization dose (per bird); b, EtMZ parental immunization dose (per bird); c, subunit vaccine immunization dose (0.1 mL / bird).
[0123] (2) Serum antibody levels: Blood samples were collected from each group 7 days after each immunization, and serum was analyzed using ELISA to detect antibody levels. Results are as follows: Figure 4 As shown, the results indicated that VP2 antibodies were not detected in serum before and one week after the first immunization. One week after the second immunization, serum antibody levels increased significantly in both the coccidia plus subunit vaccine group and the subunit vaccine alone group. The antibody level in the EtVP2 plus subunit vaccine group was higher than that in the EtMZ plus subunit vaccine group, but the difference was not significant. One week after the third immunization, antibody levels in both the coccidia plus subunit vaccine group and the subunit vaccine alone group increased substantially, showing a significant difference compared to one week after the second immunization. The antibody level in the EtVP2 plus subunit vaccine group was significantly higher than that in the EtMZ plus subunit vaccine group (P < 0.01).
[0124] (3) Cellular immune level: Peripheral anticoagulated blood was collected one week after the third immunization and incubated with three flow cytometry antibodies: CD3 PE, CD4 FITC, and CD8 APC, followed by flow cytometry analysis. Results are as follows: Figure 5 As shown, CD4 in the subunit vaccine combined with coccidia immunization group + T lymphocytes, CD8 + The results of T lymphocyte detection were significantly higher than those of the group receiving the single immunization subunit vaccine.
[0125] (4) CD4 of EtVP2, EtVP2+rVP2, EtMZ, EtMZ+rVP2, rVP2 and blank control + / CD8 + The ratios were 2.4, 2.5, 2.25, 1.65, 1.1, and 0.42, respectively. Figure 6 The results indicate that when coccidia and subunit vaccine are immunized together, the body's T-cell immune response is significantly stronger than that of subunit vaccine alone, and the body's immune function is relatively stronger, meaning that coccidia can promote the body's cellular immune response.
[0126] The above results indicate that the recombinant coccidia obtained by the above construction method have a significant immune-enhancing effect against the VP2 subunit vaccine.
[0127] This embodiment successfully inserted the novel variant VP2 gene of infectious bursal disease virus (IBDV) into the ETH_00009555 gene of *Eimeria tenella*, and obtained a stably heritable strain. The construction method described in this invention successfully constructed and screened *Eimeria tenella* expressing the novel IBDV variant VP2 gene, and the constructed recombinant coccidia vector can be used for vaccine research, providing a new method for the prevention of IBD and *Eimeria tenella*.
[0128] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.
Claims
1. An ETH_00009555 gene editing system, characterized in that, The ETH_00009555 gene editing system includes gene editing plasmids and homologous recombination fragments; The gene editing plasmid includes sgRNA and Cas9 targeting the ETH_00009555 gene; The homologous recombination fragment includes the VP2 gene and a selection marker gene.
2. The ETH_00009555 gene editing system according to claim 1, characterized in that, The sgRNA targeting the ETH_00009555 gene includes the sequence shown in SEQ ID No.
2.
3. The ETH_00009555 gene editing system according to claim 1 or 2, characterized in that, The nucleotide sequence of the VP2 gene includes the sequence shown in SEQ ID No.
1.
4. The ETH_00009555 gene editing system according to any one of claims 1 to 3, characterized in that, The selection marker gene is the mCherry red fluorescent protein gene.
5. A recombinant coccidia vector, characterized in that, The recombinant coccidia vector contains the ETH_00009555 gene editing system as described in any one of claims 1 to 4.
6. The recombinant coccidia vector according to claim 5, characterized in that, The recombinant coccidia vector is a coccidia vector that integrates the VP2 gene and the mCherry red fluorescent protein gene into the ETH_00009555 gene after being edited by the ETH_00009555 gene editing system according to any one of claims 1 to 4.
7. A method for detecting the recombinant coccidia vector according to claim 5 or 6, characterized in that, The method includes PCR amplification detection and / or fluorescence detection.
8. The method for detecting recombinant coccidia vector according to claim 7, characterized in that, The target genes detected by the PCR amplification include the VP2 gene and / or the mCherry red fluorescent protein gene sequence. Preferably, the fluorescence detection includes detection based on the fluorescence of the recombinant coccidia vector.
9. The use of the recombinant coccidia vector according to claim 5 or 6 in the preparation of a vaccine against infectious bursal disease in chickens.
10. A coccidiosis vaccine, characterized in that, The coccidiosis vaccine comprises the recombinant coccidiosis vector and VP2 subunit vaccine as described in claim 5 or 6; the VP2 subunit vaccine comprises the IBDV mutant VP2 protein; Preferably, the VP2 subunit vaccine further includes a white oil adjuvant.