CN112522215B

CN112522215B - Recombinant orf virus lacking 008 gene and construction method thereof

Info

Publication number: CN112522215B
Application number: CN201910879817.3A
Authority: CN
Inventors: 余远迪; 许国洋; 杨柳; 张素辉
Original assignee: Chongqing Academy of Animal Sciences
Current assignee: Chongqing Academy of Animal Sciences
Priority date: 2019-09-18
Filing date: 2019-09-18
Publication date: 2023-06-02
Anticipated expiration: 2039-09-18
Also published as: CN112522215A

Abstract

The invention provides a recombinant orf virus which knocks out 008 gene encoding one of F-box-like/ANK anchoring proteins; the 008 gene has a sequence shown in SEQ ID NO. 1. The recombinant orf virus has good replicative capacity, obviously reduced toxicity and good immunogenicity, thereby providing scientific data for enriching the pathogenic mechanism of the orf virus and providing a new target for developing gene deletion attenuated vaccines of the orf virus.

Description

Recombinant orf virus lacking 008 gene and construction method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a recombinant virus of sheep infectious pus lacking 008 gene and a construction method thereof.

Background

An aphtha is an acute, highly contagious zoonotic infectious disease caused by the Orf virus (ORFV), which affects mainly goats and sheep. Clinically, virus infection in sheep is typically characterized by proliferative inflammation, manifested by the onset of erythema at the sheep's lips, tongue, nose, udder, etc., followed by the formation of papules, vesicles, yellow creamy pustules, and crusting, which eventually become dry, and the virus can repeatedly infect the host. In the infection process of the aphtha virus, the immune system of the host plays an important role, CD4+ T cells, interferon and the like generated in the host can inhibit the replication of the virus, but the virus cannot be prevented from repeatedly infecting the host, so that the aphtha virus has important significance in regulating the immunity of the host by utilizing the strategy that the aphtha virus escapes from the host to immunize by utilizing the evolution of some virulence factors existing in the aphtha virus through the interaction of the aphtha virus and the host.

The orf virus belongs to parapoxvirus genus of poxviridae, is linear double-stranded DNA virus, has genome size of 134-139kb, has large central coding region in the middle of the genome, and participates in virus assembly and release; the inverted terminal repeats are at both ends (ORF 001-ORF008, ORF112-ORF 134). The virulence genes, the coding genes related to host tropism and the immunoregulatory genes of viruses are located in the terminal regions on both sides of the genome, so that the terminal genes of the orf viruses are not completely annotated at present, and the functions of some important proteins are further discovered and verified, so that the replication and infection mechanisms of the orf viruses and the relation between the viruses and the hosts are clarified.

Anchor protein repeats and F-box domain proteins have been shown to be widely present in poxviruses, ranging in size from 400 to 650 amino acids, with 5-10 ANK repeats at the N-terminus and conserved sequences similar to the F-box domain at the C-terminus, with recruitment of substrates to the ubiquitin ligase complex of cell SCF (SKP-1, cullin, F-box) (Sonnberg et al.; herbert et al, 2015). The orf virus F-box-like anchor protein ORFV008 is functionally related to the SCF ubiquitin ligase complex and is expressed early in the viral infection host and plays a role in viral replication capacity and pathogenicity.

The regularly-spaced clustered short palindromic repeats/Cas protein (CRISPR/Cas 9) system is a natural microbial immune mechanism against the invasion of other pathogens. The system consists of RNA-guided Cas9 endonuclease (from streptococcus pyogenes (Streptococcus pyogenes), single guide RNA (sgRNA) and trans-activation CRISPR RNA (tracrrRNA) which have genome editing suitable for eukaryotic cells, can be pulled to the vicinity of target genes by the sgRNA, recognizes the target gene's PAM sequence NGG (N is A, T, C, G) and cleaves at the position of 3-4 bases on the PAM sequence, destroys the target gene sequence, triggers the repair mechanism of DNA inside the cells, one of the repair modes is mutation, deletion and insertion of bases formed at the junction, thereby deleting the target gene, and the other is homologous recombination repair mode, the former is dominant.

Disclosure of Invention

The invention aims to provide a recombinant orf virus, which does not influence the in vitro replication capacity of the orf virus, can obviously reduce the virulence of the virus, keeps good immunogenicity, and can be used as a new scheme of an orf virus attenuated vaccine.

The aim of the invention is achieved by the following measures:

a recombinant orf virus that knocks out 008 gene encoding one of the F-box-like/ANK anchor proteins.

The sequence of the 008 gene is shown as SEQ ID NO. 1.

Another object of the present invention is to provide a method for preparing the recombinant orf virus.

The preparation method of the recombinant orf virus comprises the step of knocking out 008 genes of the orf virus by adopting CRISPR/Cas9 plasmid, wherein primers of the plasmid are as follows:

Oligo1-F：5’-ACCGCGTCAGTTGCCACGAGCGCG-3’

Oligo1-R：5’-AAACCGCGCTCGTGGCAACTGACGC-3’

Oligo2-F：5’-ACCGCATCACGTCCACGAGCGCGG-3’

Oligo2-R：5’-AAACCCGCGCTCGTGGACGTGATGC-3’。

specifically, the preparation method of the recombinant virus for the aphtha comprises the following steps:

(1) pUgRV-ORFV008-1 and pUgRV-ORFV008-2 plasmids with the gene sequences of the orf viruses were obtained: taking the sequence of the gene 008 of the strain of the orf virus as a template, adding ACCG at the 5 'end of a primer coding chain, adding AAAC at the 5' end of a non-coding chain template, designing two pairs of primers of the 008 gene, and carrying out in vitro annealing to form double chains, and connecting with UgRV vectors subjected to Bbs I digestion to obtain pUgRV-ORFV008-1 and pUgRV-ORFV008-2 plasmids;

(2) Purification of viral genome: extracting viral genome DNA and purifying by ethanol precipitation, and identifying the genomic DNA by PCR amplification of the conserved gene F1L of the orf virus;

(3) Rescue of recombinant virus: the plasmids pUgRV-ORFV008-1, pUgRV-ORFV008-2 and empty plasmid are extracted, LT cells are co-transfected with viral genome, supernatant containing the virus is collected, repeated plaque screening and purification are carried out, and the P3 generation recombinant orf virus is obtained and named rORFV008-mut.

Advantageous effects

1. The obtained orf virus has high virus proliferation titer, good stability, obviously reduced virus virulence, high safety, strong immunogenicity, and similar immunization and intensity infection to the parent strain, and the organism can generate immune response to various antigens of the virus, thus being applicable to local inoculation and inducing mucosal immunity. Provides a new direction and thought for developing and producing the weak vaccine of the orf virus.

2. The genome of the orf virus has large capacity, the replication capacity of the virus is not affected by the deletion of 008 gene locus, and a plurality of exogenous genes can be inserted into the locus to construct the recombinant orf virus for expressing other exogenous genes.

3. Because the framework genome of the orf virus is huge (about 134 kb), and the GC content of the genome is up to 63% -64%, if the target gene is deleted by a conventional homologous recombination method, the success rate is low. The invention adopts a specific CRISPR/Cas9 gene editing technology, can greatly improve DNA repair probability in cells by destroying target gene sequences, increases recombination probability, is easy to screen recombinant viruses, and simply and efficiently obtains recombinant orf viruses with 008 genes deleted.

4. The invention designs and synthesizes 2 gRNA sequences capable of specifically recognizing 008 genes, constructs CRISPR/Cas9 plasmid knockout orf virus 008 genes, saves recombinant viruses and carries out sequencing identification. The virus is used for infecting goat mucosa and body surface skin, the formation of body surface focus is dynamically observed, the pathology of skin biopsy is detected, the neutralizing titer of antibodies in serum is detected, and the toxicity and immunogenicity of recombinant aphtha virus lacking 008 genes are evaluated.

Drawings

FIG. 1 UgRV (CRISPR vector) plasmid insertion of the Douglas 008 Gene schematic

FIG. 2 shows a diagram of the cleavage results of UgRV plasmid vector Bbs I (M: marker;1: cleavage identification result);

FIG. 3 shows a pUgRV-ORFV008-1 plasmid sequencing identification map;

FIG. 4 shows a pUgRV-ORFV008-2 plasmid sequencing identification map;

FIG. 5 shows an identification of the orf virus (M: marker;1: isolated virus identification results)

FIG. 6 is a diagram of P2-generation virus infected cells, wherein FIG. a is a diagram of P2-generation cytopathic infection, and FIG. b is a blank control;

FIG. 7 is a PCR identification P2 generation virus electrophoresis chart (M: marker;1: P2 generation recombinant virus PCR product);

FIG. 8 is a diagram of P3-generation virus infected cells, wherein FIG. a is a diagram of P2-generation cytopathic infection, and FIG. b is a blank control;

FIG. 9 is an electrophoretogram of PCR identification of the P3 generation virus;

FIG. 10 is a sequencing alignment of recombinant viruses;

FIG. 11 shows the detection of virus proliferation potency.

Detailed Description

The invention will be described in detail below with reference to the drawings and the detailed description.

The nucleotide sequence of the gene 008 of the orf virus is shown as SEQ ID NO. 1.

1. Obtaining the aphtha virus lacking 008 genes:

1) Taking an orf virus strain 008 gene sequence as a template, adding ACCG at the 5 'end of a primer coding chain, adding AAAC at the 5' end of a non-coding chain template, wherein the primer is as follows:

the strains of the aphtha virus are derived from commercial strains of the group of the OVs OV-SA00 (NCBI: AY 386264.1), OV-IA82 (NCBI: AY 386263.1), NZ2 (DQ 184476.1), D1701 (HM 133903.1) aphtha virus or isolated strains of the aphtha virus obtained clinically from goats in Chongqing. The method comprises the following specific steps:

step 1: cloning of the gRNA of 008 gene onto CRISPR vector (fig. 1): the two pairs of primers were annealed in vitro to form a double strand, and ligated with UgRV vector digested with Bbs I (FIG. 2) to obtain pUgRV-ORFV008-1 and pUgRV-ORFV008-2 plasmids, and the sequencing results were identical with the primer sequences (FIG. 3, FIG. 4).

Step 2, purification of viral genome: the viral genomic DNA was extracted and purified by ethanol precipitation, and the genomic DNA was identified by PCR (primers for amplifying F1L gene: F1L-F:5'-ATGGATCCGCCGGAAATTAC-3'; F1L-R: 5'-AACAATGGCGGTAACCAGCA-3') using the conserved gene F1L of the orf virus (FIG. 5).

Step 3 rescue of recombinant virus: the endotoxin-removed plasmids pUgRV-ORFV008-1, pUgRV-ORFV008-2 and empty plasmid were extracted, LT cells were co-transfected with viral genome, and after reaching 70-80% of cytopathy, the cell plates were repeatedly freeze-thawed 3 times, centrifuged at 3000rpm for 5 minutes, and the virus-containing supernatant was collected. LT cells are paved on a 6-hole cell culture plate, virus supernatant is inoculated into cell holes, after 72 hours, single plaque is picked up, added into 500 mu L of DMEM, and repeatedly blown and dissolved to obtain the supernatant virus liquid. The 50. Mu.L of LT cells spread on a 12-well plate were inoculated, and before all the cells were diseased but not shed (FIG. 6), the cell supernatant was collected, the viral genome was extracted, the F1L gene primer of step 2 was used for PCR identification, and the amplified product was detected by 1% agarose gel electrophoresis to see a band of about 1000bp in size (FIG. 7). The virus solution identified as positive was purified for the P2 generation and the next round of plaque screening was performed for the P3 generation (fig. 8, fig. 9). Sequencing results showed that 008 gene was knocked out in the recombinant virus (FIG. 10), which was named rORFV008-mut.

TABLE 1 rescue of recombinant viruses

2. Virus replication curve for detecting virus replication capacity

The cells of fetal sheep turbinate (OFTu) are paved on a 12-hole cell plate, after the cell density reaches 50-60%, the virus parent strain ORFV-WT and the recombinant orf virus rORFV-mut lacking 008 genes are respectively inoculated at the dose of a complex number of 0.01MOI, incubated at 37 ℃, gently rocked once every 15min, incubated for 1h, the virus liquid is discarded, PBS is used for washing the cells twice, and DMEM containing 0.2% BSA is added as a maintenance liquid. Supernatants were taken 24, 36, 48, 60 and 72 hours post-viral infection, frozen at-80℃and assayed for TCID on MDBK cells ₅₀ Curves were plotted, and three replicates were made for each time point. The results showed no difference in the proliferation capacity of the two viruses (FIG. 11), indicating that the aphtha virus lacking 008 gene still has good replication capacity.

3. The virulence of the aphtha virus lacking 008 genes is obviously reduced, and the aphtha virus has stronger immunogenicity.

Healthy 3-5 month old lambs without infection by the aphtha virus and without immunization with the aphtha vaccine were randomly divided into three groups of three lambs each (three for the control vaccinated group, three for the ORFV-WT vaccinated group, and three for the rORFV008-mut vaccinated group). The lambs were sedated with the safety drug and the vaccinated sites were rinsed with sterile aqueous physiological saline. Score lines along 2cm and 5cm on the right labial mucosa, right dorsal and right hind limb strand inner side of the test animals, respectively, and were 10-fold ⁷ TCID50/ml virus (0.5 ml) was inoculated with each site using a cotton swab and the control group was PBS as a control.

(1) After inoculation, the lesion formation is observed and evaluated day by day, the body temperature is detected at regular time, the inoculation time is 0d, and 21d is observed. The criteria evaluated were the extent of erythema, papules, pustules and adherent crusting, each scored according to the width of lesions along the score line: 1 min, lesions <0.5cm;2 minutes, 0.5cm < lesion < cm; 3 minutes, lesions are more than 1 cm, the total daily score of each small sheep is the sum of three lesion types, the lesions are scored according to the width of the lesions in the observation process, and compared with a parent strain group, the rORFV008-mut inoculation group is reduced by 30% -66%.

(2) Skin biopsies were collected on days 2, 5, 8, 12 and 21, fixed with 10% buffered formalin, paraffin embedded, sectioned, stained with hematoxylin and eosin standard methods, and microscopic observations of the sections revealed that: compared with the inoculated parent strain group, the rORFV008-mut inoculated group has no obvious lesion, which indicates that the toxicity of the recombinant virus is obviously reduced.

(3) After vaccination, body temperature was checked at regular intervals and vaccinated again after 4 weeks, as observed daily. The neutralizing titer of the antibodies was detected by sheep serum at weeks 3, 4, 6 and 8, respectively, the antibody titers of the parental strain group seed group and the rORFV008-mut seed group were significantly higher than those of the control group, but there was no difference between the two seed groups, indicating that the aphtha virus lacking 008 gene still had good immunogenicity.

<110> Chongqing City academy of livestock sciences

<120> recombinant orf virus lacking 008 gene and method for constructing the same

<160>

<210> 1

<211> 1551

<212> DNA

<213> Artificial (Artificial sequence)

<400> 1

atgctctcgc gggagtccgt cgtggctccg cacgcggacc tgctcttccg ctacctggag 60

tccgggcagg tggatctcgc cacggtccgc gcgctcgtgg caactgacgc ggacgtgaac 120

ttccgcggcg agtacgggcg cacgccgctg cacctctgcg tgcacttcgc gcggcacgag 180

cagtgtgcgg agatcgtgcg cgtgctgctg gaggctggcg cggacgtaaa cgccaaggac 240

acctgcggct tcacgccgct gcacgcctac gtgcagcacg actgcgtgcg gccggaggtg 300

gtcgcgctca tgctggaggc gggcgcagac gtggtctgcg acgacagctt cgtcttctac 360

gacagcgcgc tctcctcctt cctgtcttcc tgcggctccg acggcaccga gctcgaggtc 420

gcgcggctgc tgctggacgc gggcgcgcgc gtgaacgagg gcgacaccta cggcatgacg 480

ccgctgcacg tgtacgccaa gaaccagtgg atccgcgagg acgtgctgcg gctgctgctc 540

gagcgcggcg cgaacccaaa cgcctgcgac tgccacggcg tgacgccgct ggcggcgctg 600

ctgggctccg gcggcgtctc cgccgcgctc gtggacgtga tgctgcgcgc gggcgcagac 660

gcacgcgccg tggacgcata caggcgcacg acactgcacc acctcgcgcg cacggccaag 720

atctccgagg gcctggtgcg catgctcacg ggcctgggcg tggacccggc cgcggtagac 780

gcgagtggga acaccatgct gcactacatg gcgacctacg ggcgctgcgc tcgcggcgtc 840

gtggaattcg tgctcgagcg cgggctggac ctgaacctgc gcaacaacaa cctgcagacc 900

gcgctgcacc gcgcggcggt gttcagccac ggcgcctgct gccggctggt gcgcatgggc 960

gcggagctcg ggcacgtggc ggcctcgggc ctatgcgcgg tctctgagat gctgcgccgc 1020

aacaacgtgc gtgcgacggc cgccgtgctc gcgcgccggc cgccgacgga actgctcgtg 1080

cgcgcgctgc tcacatccga gcgctggggc cacgtgttca aacgctcgga ggccgcgctg 1140

ctgtgcgtgc aggagctggc gctgcgcggc gagggcgcgc gcctgctggc ggagcgagcg 1200

ctggcggact acgcgaccgt ggtccgcgcg tgcgagcagg agatcgcgag catgcgcgcg 1260

gtgcgctgcc acacggacgc gacgctgctg gacgtgctgc gcgcggcgca cgacgcgaag 1320

gcgctcttcg tgtcgaacgc cttcctggag cgcgcggccg agttccccat ctacgggacg 1380

gcgctcttcg gcaaggtctg catgatgcgg ctgcgcgtct cgctggctga gcagatcgcc 1440

ggcctcatgt gcccgtgcgc cctgccgccg gagatcgtga cctccatcct gtgcttcctg 1500

ccgtacgagt cgctgctgga cctgcgccgc gccatgctga cccgcccctg a 1551

Claims

1. A recombinant orf virus, characterized in that: the 008 gene with the sequence shown as SEQ ID NO.1 is knocked out.