CN114807059A - Novel PCV3 virus strain and PCV3 genetic engineering subunit vaccine - Google Patents

Abstract

The invention discloses a novel PCV3 virus strain and PCV3 genetic engineering subunit vaccine, wherein the virus strain contains Cap protein with an amino acid sequence shown as SEQ ID NO. 1, and is preserved in China center for type culture Collection with the preservation number of CCTCC NO. V202208. The PCV3 genetic engineering subunit vaccine is prepared by inactivating Cap protein by a hydrolyzable inactivator, and then adding an aqueous adjuvant for emulsification, and can be used for PCV3 emergency immune evaluation. Experiments show that the PCV3 nucleic acid positive rate is reduced from 100% to 10% before and after the subunit vaccine is applied, the ELISA antibody positive rate is improved from 20% in the initial stage of natural infection to 80% after vaccine immunization, and the clinical application effect is good.

Description

Novel PCV3 virus strain and PCV3 genetic engineering subunit vaccine

Technical Field

The invention relates to the technical field of biological products for livestock, in particular to a novel PCV3 virus strain and a PCV3 genetic engineering subunit vaccine prepared based on the virus strain.

Background

Porcine Circovirus (PCV) currently has four established genotypes: PCV1, PCV2, PCV3 and PCV 4. PCV1 is a non-pathogenic virus, the latter three being all pathogenic viruses.

PCV3 was first discovered and reported by Palinski et al from a dead fetus sample of a certain pig farm sow with unexplained dermatitis syndrome and abortion by means of metagenome sequencing, and was named as porcine circovirus type 3 (PCV 3). The prevalence of PCV3 in pig farms was subsequently reported in most pig-raising countries of the world. Since PCV3 was discovered in the united states, china, korea, polish, brazil and italy have also reported the presence of the virus in succession. Pigs infected with PCV3 show Porcine Dermatitis and Nephrotic Syndrome (PDNS), reproductive disorders, respiratory, gastrointestinal and nervous system diseases, multiple system inflammation, myocarditis, etc. PCV3 genome has been detected by PCR methods in oral fluid and nasal swabs as well as feces, semen and colostrum, horizontal transmission by direct contact may be the main transmission pathway. The pathogenicity and immunological mechanisms of PCV3 are currently unknown, its high incidence may pose a potential threat to the swine industry worldwide, and the widespread presence of PCV3 has raised high concern to the swine industry in countries worldwide.

Since PCV3 strain is difficult to separate on cells, the development of in vitro differential diagnosis method aiming at PCV3 is greatly limited, and the prevention and treatment of PCV3 virus also have certain difficulty.

Disclosure of Invention

The invention aims to provide a novel porcine circovirus type 3 (PCV3) strain and a genetic engineering subunit vaccine based on the strain, thereby making up the defects of the prior art.

The technical scheme of the invention is detailed as follows:

in a first aspect, the invention provides a novel PCV3 virus strain, which contains Cap protein with an amino acid sequence shown as SEQ ID NO. 1, and is preserved in China center for type culture Collection with the preservation number of CCTCC NO. V202208.

In a second aspect, the present invention provides the use of the above PCV3 virus strain for the preparation of a PCV3 vaccine.

In a third aspect, the invention provides a PCV3 genetic engineering subunit vaccine, which is prepared from Cap protein with an amino acid sequence shown in SEQ ID NO. 1.

Alternatively or preferably, the vaccine is prepared by inactivating Cap protein with a hydrolyzable fire extinguishing agent, and adding an aqueous adjuvant for emulsification.

Alternatively or preferably, in the vaccine, the mass ratio of the Cap protein to the aqueous adjuvant is 8: 2.

Alternatively or preferably, the content of Cap protein in the vaccine is not less than 50. mu.g/mL.

Alternatively or preferably, in the vaccine, the Cap protein is prepared by the following method:

the virus strain of claim 1 is used as a template, SEQ ID NO 3-4 is used as a primer for PCR amplification, and an amplification product is inserted into an expression vector PET-28a (+) to construct an expression recombinant plasmid PET-ORF 2;

PET-ORF2 is transformed into escherichia coli BL21 host bacteria, and His-Cap recombinant fusion protein is expressed under IPTG induction;

and purifying the recombinant fusion protein by using a purification column aiming at the His (histidine) tag to obtain the Cap protein with the His tag. .

Description of the drawings:

PET-28a (+): an expression carrier contains kanamycin-resistant gene, the corresponding expression host bacterium is colibacillus and is used for expressing target gene, the genome contains protein labels N-His, N-T7 or C-His, and IPTG or lactose and the like can be used for induction expression.

Escherichia coli BL 21: a host bacterium for broad protein expression, also known as Escherichia coli BL21(DE3) bacterium. The chromosome carries a T7 RNA polymerase gene controlled by a lacUV5 promoter, and the exogenous target gene driven by a T7 promoter can be efficiently expressed under IPTG induction.

IPTG: isopropyl-beta-D-thiogalactoside of the formula C ₉ H18O ₅ S, can cause the transcription process of the lactose operon, and can induce the downstream gene of the lactose operon to correspond to the eggExpression of white.

An aqueous adjuvant: the main functions are to adsorb antigen, make the antigen slowly release in vivo, enhance humoral immunity, help to improve antigen presentation efficiency, stimulate immune cells to rapidly proliferate. Commercially available products can be purchased directly. Some water-based adjuvants can be directly and uniformly mixed with antigen without emulsification.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a novel PCV3 virus strain PCV3 LY strain, which has 87.08-87.85% of gene sequence homology and 90.70-93.49% of amino acid sequence homology with the disclosed PCV3 virus. A genetic engineering subunit vaccine is prepared by using Cap protein of the virus strain, can be used for PCV3 emergency immune evaluation, and is prepared by injecting and immunizing 4-6-week-old weaned piglets which are positively infected by PCV3 twice. The PCV3 nucleic acid positive rate before and after the subunit vaccine is applied is reduced from 100% to 10%, the ELISA antibody positive rate is improved from 20% in the initial stage of natural infection to 80% after vaccine immunization, and the clinical application effect is good.

Preservation information:

name: porcine circovirus type 3PCV3 LY strain (PCV3 LY strain);

the preservation unit: china center for type culture Collection;

and (4) storage address: wuhan, Wuhan university, post 430072, China;

the preservation number is: CCTCC No. v202208;

preservation time: 3 months and 10 days in 2022.

Drawings

FIG. 1 shows the nucleic acid electrophoresis of PCV3 ORF2 gene, M:5000 Marker; 1. a gene of interest; 2. negative water control.

FIG. 2 is SDS-PAGE electrophoresis of expression PCV3 Cap protein, 1. expression protein induced by whole engineering bacteria (unpurified); 2. the engineering bacteria do not induce to express protein; 3. and (5) purifying the target protein.

Detailed Description

The present invention will be described in detail with reference to the preferred embodiments, and the chemicals or reagents used in the present invention may be selected from other existing products according to their functions, and are not limited to the specific descriptions of the embodiments. For experiments where no specific conditions are noted in the examples, it is generally possible to operate under conventional conditions, such as those described in the molecular cloning instructions written by J.Sambruke (Sambrook), et al, or according to the manufacturer's recommendations.

Example 1 isolation and identification of PCV3 LY Strain

1.1 onset of disease

The piglets in a certain pig farm in Liaoyang city of Liaoning show symptoms of slow growth, gradual emaciation, cough, dyspnea, no spirit, pale skin, jaundice, diarrhea, swollen lymph nodes (which can be felt by touching femoral sulcus lymph nodes), disharmony of hair and the like 2-3 days or about one week after weaning, the morbidity is 20% -60%, and the mortality is 10% -40%.

1.2 clinical anatomical conditions

The dead pigs with the anatomical diseases can be seen in large, hyperemia and congestion in lymph nodes of tissues such as mesentery, inguinal and the like, lung hyperemia and congestion, kidney hyperemia and congestion, plaques and various pathological changes with different degrees.

1.3 laboratory diagnostics

1.3.1 treatment of pathogens and tissue Virus Total DNA/RNA extraction

According to clinical manifestations and autopsy lesions of the pigs died of diseases, weaned pig exhaustion syndrome (PMWS) caused by porcine circovirus infection is suspected preliminarily, so that lung tissues of the dead pigs are taken aseptically, the tissues of the lung tissues of the dead pigs are crushed and homogenized by a tissue triturator, viral total nucleic acid in the tissues is extracted by a viral total DNA/RNA extraction kit, and the concentration of the nucleic acid is determined to be 396 ng/uL.

1.3.2 identification of viruses by PCR

Individual colonies were PCR amplified using primers designed to detect PCV2 and PCV3 (see Table 1 for details of upstream and downstream primer sequences) and electrophoresed in a 1% agarose gel.

TABLE 1 primer sequence Listing

1.3.3 Gene sequencing

The result of nucleic acid electrophoresis shows that a specific target band appears in an amplified lane of PCV3, which is shown in detail in FIG. 1. Cutting off the target band and sending to Shanghai worker for sequencing. The sequencing result is found to be a new gene sequence of PCV3 through BLAST analysis, the homology with the gene sequence of the known PCV3 strain is about 87.08-87.85%, the homology with the amino acid sequence of the known PCV3 strain is 90.70-93.49%, and the homology with the genome of other PCV (PCV2/PCV4) is 43.2-51.5%.

In order to exclude the gene due to the error generated in the amplification process, multiple amplifications and sequencing were performed, and the sequencing result showed that the nucleotide sequence was shown as SEQ ID NO. 2. The PCV3 ORF2 gene amplified in the tissue disease material is proved to be a new gene, is a variant strain of the current epidemic strain and is named as PCV3 LY strain. The strain is preserved in China center for type culture Collection in Wuhan, China with the preservation number of CCTCC NO: V202208.

Example 2 preparation of core antigen Cap protein of PCV3 genetically engineered subunit vaccine

2.1 construction and identification of genetically engineered Escherichia coli containing PCV3 ORF2 novel gene

According to a sequence at two ends of a new gene of PCV3 ORF2 and a polyclonal enzyme cutting site of an expression vector PET-28a, specific amplification primers are designed as follows:

PCV3 New ORF 2-F: 5'-GGG GGA TCC ATG AGA CAC AGA GCT ATA TTC-3' (BamHI sites added),

PCV3 novel ORF 2-R: 5'-GGG AAG CTT TTA GAG AAC GGA CTT GTA ACG-3' (HindIII sites added).

The primers were synthesized by Shanghai Biotechnology engineering services, Inc. PCR amplification was carried out using DNA extracted from the tissue virus solution of PCV3 LY strain (CCTCC NO: V202208) of example 1 as a template.

The amplified product is target gene sequence of 645bp, and the target gene is recovered by DNA purification and recovery kit. The target gene and a carrier PET-28a (+) are subjected to double enzyme digestion and connected to obtain a PET-ORF2 recombinant plasmid, the recombinant plasmid is transferred into an expressive escherichia coli BL21 host bacterium according to a conventional method, a single clone is selected, a positive clone is identified by an enzyme digestion method, bacteria are shaken and sequenced, and the sequencing result is shown in SEQ ID NO. 2, and the amino acid sequence of the translated protein is SEQ ID NO. 1.

2.2 preparation of novel PCV3 Cap protein

Inoculating expressive BL21 host bacteria transformed by single clone recombinant plasmid PET-ORF2 with correct sequencing result into 5mL LB bacterial culture medium containing ampicillin (100mg/L), shaking and culturing overnight at 37 ℃, taking out 1mL of culture next day, inoculating the culture into 120mL of LB culture medium, shaking and culturing for 2.5h at 37 ℃, shaking the bacterial liquid to be in a semitransparent semi-turbid state, measuring OD600 to be 0.6-0.8 (logarithmic growth phase) by an absorptiometry, taking 10mL of the culture before induction as a control before induction, changing the shaking temperature to 30 ℃, adding IPTG (final concentration is 1.0mmol/L) for induction, collecting 10mL of the bacterial liquid after 4h induction, taking the bacterial liquid of the induced non-transformed PET vector as a blank control, and taking the bacterial liquid of the induced transformed PET vector as an empty vector control. Taking out the bacteria liquid for induction expression, centrifuging to remove supernatant, then resuspending the bacteria by 0.5mL of 1 XPBS, ultrasonically cracking and crushing the bacteria, adding 2 Xsample Buffer according to a ratio of 1:1, boiling for 10min, centrifuging to take supernatant, and carrying out electrophoretic identification and analysis on the supernatant by 12% SDS-PAGE to obtain the target protein of 27-28 kDa, which is detailed in figure 2. And purifying the target protein by using a His label purification kit according to specified operation, wherein the purity of the purified His-Cap protein is more than 95%.

Example 3 preparation and safety test of PCV3 genetic engineering subunit vaccine

The PCV3 Cap protein purified in example 2 was dissolved in sterile physiological saline, the obtained solution was filtered through a 0.22 μm sterile filter to obtain a sterile protein solution, and the concentration of the filtered sterile protein solution was measured by spectrophotometry, and the protein concentration was 1.05 mg/mL.

Diluting the protein solution with sterile normal saline to a final concentration of not less than 62.5 mug/mL, and mixing with a sterilized water adjuvant according to a volume ratio of 8:2, preparing the vaccine, and emulsifying and mixing the mixture uniformly by adopting a sterile magnetic stirrer to prepare the PCV3 genetic engineering subunit vaccine preparation, wherein the finished product is equivalent to that each milliliter of the vaccine preparation contains no less than 50 mu g of Cap protein.

The PCV3 gene engineering subunit vaccine preparation is used for carrying out safety inspection on mice according to the dose of 1.0mL per mouse, and is respectively injected with 0.5mL in an intraperitoneal way and 0.5mL in a cervical and dorsal subcutaneous way, and is continuously observed for 7 days. The observation results show that all the mice injected with the PCV3 gene engineering subunit vaccine have good spirit, good food intake and drinking water, good production and development and healthy activity, and the safety inspection is qualified.

Example 4 application of PCV3 gene engineering subunit vaccine in emergency immune evaluation of clinical PCV3 infection morbidity pig farm

The PCV3 genetic engineering subunit vaccine qualified in laboratory security inspection is used for an emergency immune evaluation application test of a clinical PCV3 infection morbidity. The application method of the emergency immune evaluation comprises the following steps: randomly selecting 100 weaned piglets with positive infection detected by PCV3 and 4-6 weeks old, and injecting PCV3 gene engineering subunit vaccine preparation into neck muscle according to the dose of 2.0mL per head for immunization.

And carrying out second injection immunization at the same dose 14 days after the first immunization, collecting blood of all test pigs 21 days after the second immunization, separating serum, and respectively extracting nucleic acid for PCR detection and serum antibody level ELISA detection.

The application result shows that the PCV3 nucleic acid positive rate before the subunit vaccine is applied is 100% (100/100), the nucleic acid detection positive rate after the subunit vaccine is applied is 10% (10/100), the ELISA antibody positive rate is improved from 20% (20/100) in the initial stage of natural infection to 80% (80/100) after vaccine immunization, and the clinical application effect shows good.

The inventive concept is explained in detail herein using specific examples, which are given only to aid in understanding the core concepts of the invention. It should be understood that any obvious modifications, equivalents and other improvements made by those skilled in the art without departing from the spirit of the present invention are included in the scope of the present invention.

Sequence listing

<110> Shandong Xin De science and technology Co., Ltd

<120> novel PCV3 virus strain and PCV3 genetic engineering subunit vaccine

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 214

<212> PRT

<213> Porcine circovirus (Portone circovirus)

<400> 1

Met Arg His Arg Leu Val Thr Asn Pro Asn Phe Phe Gly Ala Val Glu

1 5 10 15

Val Ala Met Ser Asp Ile His Val Asp Thr Tyr Tyr Thr Lys Gln Cys

20 25 30

Ser Pro Leu Asn Gly Gly Val Ile Cys Val Gln Pro Trp Gly Gly Ser

35 40 45

Gly Glu Val Glu Glu Ala Leu Ser Trp Val Ser Ala Gly Ser Ser Arg

50 55 60

Gln Asn Trp Phe Gly Gly Glu Val Thr Ala Val Ile Ser Phe Glu Tyr

65 70 75 80

Tyr Lys Ile Lys Asp Glu Ser Tyr Gln Thr Lys Thr Met Phe Gly Pro

85 90 95

Arg Ala Ile Asp Leu Asp Gly Ala Trp Thr Thr Asn Thr Trp Pro Glu

100 105 110

His Ser Phe Cys Leu Leu Ser Arg Arg Asn Tyr Arg Ala Glu Cys Asn

115 120 125

Phe His Leu Arg Tyr Leu Ile Ile Phe Lys Ala Asn Gly Ser Phe Pro

130 135 140

Phe Val Pro Ala Gly Asn Glu Val Val Gly Val Pro Gly Leu Val Ile

145 150 155 160

Leu Arg Gly Ser Asn Gly Asn Asp Val His Gly Gly Val Phe Leu Cys

165 170 175

Val Val Cys Ala Ser Cys Gly Pro Pro Asn Glu Ser Phe Ser Ser Asp

180 185 190

Ile Ala Pro Ser Val Ala Ser Ser Ser Pro Trp Ala Gly Ser Ser Ser

195 200 205

Glu Tyr Ser Ser Val Ser

210

<210> 2

<211> 645

<212> DNA

<213> Porcine circovirus (Portone circovirus)

<400> 2

atgagacaca gacttgtaac gaatccaaac ttctttggtg ccgtagaagt cgctatgtca 60

gatattcatg tcgacacata ctacacaaag cagtgctccc cattgaacgg tggggtcata 120

tgtgttcagc catggggtgg gtctggagaa gttgaagagg ctttgtcctg ggtgagcgct 180

ggtagttccc gccagaattg gtttgggggt gaagtaacgg ctgtgattag ctttgaatat 240

tataagatta aagatgaaag ttaccaaaca aaaactatgt tcgggccaag agccatagat 300

ctagacggcg cctggaccac aaacacttgg cccgaacata gtttttgttt gctgagccgg 360

agaaattaca gggctgagtg taactttcat cttcgttatc ttataatatt caaagctaat 420

ggcagtttcc cattcgttcc ggcgggtaat gaagtggttg gcgtgccagg gcttgttatt 480

ctgaggggtt ccaacggaaa tgacgttcat ggtggagtat ttctttgtgt agtatgtgcc 540

agctgtgggc ctcctaatga aagcttttct tctgacatag cgccttctgt ggcgtcgtcg 600

tctccttggg cggggtcttc ttctgaatat agctctgtgt cttaa 645

Claims (7)

1. The novel PCV3 virus strain is characterized by comprising Cap protein with an amino acid sequence shown as SEQ ID NO. 1, and being preserved in China center for type culture Collection with the preservation number of CCTCC NO. V202208.

2. Use of the PCV3 virus strain according to claim 1 for the preparation of a PCV3 vaccine.

3. A PCV3 genetic engineering subunit vaccine is characterized in that the vaccine is prepared by Cap protein with an amino acid sequence shown as SEQ ID NO. 1.

4. The vaccine of claim 3, wherein the Cap protein is prepared by inactivating a hydrolyzable fire extinguishing agent and then emulsifying the inactivated Cap protein with an aqueous adjuvant.

5. The vaccine of claim 4, wherein the mass ratio of Cap protein to aqueous adjuvant is 8: 2.

6. The vaccine of claim 4, wherein the Cap protein is present in an amount of no less than 50 μ g/mL.

7. The vaccine of claim 4, wherein the Cap protein is prepared by the following method:

the virus strain of claim 1 is used as a template, SEQ ID NO 3-4 is used as a primer for PCR amplification, and an amplification product is inserted into an expression vector PET-28a (+) to construct an expression recombinant plasmid PET-ORF 2;

PET-ORF2 is transformed into escherichia coli BL21 host bacteria, and His-Cap recombinant fusion protein is expressed under IPTG induction;

and purifying the recombinant fusion protein by adopting a purification column aiming at the His tag to obtain the Cap protein with the His tag.

Images