TWI401319B - Porcine circovirus type 2 capsid protein subunits and the use thereof - Google Patents

Description

Porcine circovirus type 2 coat protein subunit fragment and use thereof

The invention utilizes an E. coli expression system to prepare a subunit fragment of a Porcine circovirus type 2 (PCV2) capsid protein (Cap) with good specificity and sensitivity, and uses the same to diagnose a pig In vivo PCV2 virus infection. The invention further provides a serum diagnostic method and kit for detecting PCV2 infection in a pig.

The porcine circovirus (PCV) belongs to the circoviridae family ( Circoviridae ). The virion has a diameter of about 17 nm and has a single-stranded circular DNA gene, which is about 1.76 kb long and contains two major open reading frames ORF1. ORF2, which can transcribe and translate replication associated proteins and capsid proteins, respectively (see, Allan et al, Vet. Microbiol. 44(1): 49-64, 1995; and Nawagitgul et al, J Gen. Virol. 81(9): 2281-2287, 2000). The first type of porcine circovirus (PCV1) was first isolated from the PK-15 cell line in 1974 and is now confirmed to be ubiquitous in pig tissues but not pathogenic (Tisher et al., Nature 295:64- 66, 1982). Recently, the newly discovered type 2 virus strain (PCV2) was isolated in the lymph nodes of pigs exhibiting post-weaning multisystemic wasting syndrome (PMWS) symptoms, and was found by nucleic acid analysis. The novel virus has 60-75% homology with PCV1. This disease mainly occurs in pigs aged 6-12 weeks. Clinically, symptoms such as progressive wasting, depression, diarrhea, and jaundice can be seen. Interstitial pneumonia, lymphoid follicle loss, and occasional hepatitis and nephritis can be seen on the lesion. Neurological symptoms, etc. (Krakowka et al., Vet. Pathol. 37(3): 254-63, 2000; and Morozov et al., J. Clin. Microbiol. 36(9): 2535-2541, 1998), and if small and small When a virus (parvovirus) or PRRS virus is co-infected, more pronounced clinical symptoms are produced (see, for example, Allan et al., Vet. Microbiol. 66(2): 115-23, 1999; and Meehan et al., Arch. Virol. 46(4): 835-842, 2001).

According to previous research, PCV2 has been commonly infected with most pigs in the world (Allan and Eills, J. Vet. Diagn. Invest. 12(1): 3-14, 2000); and in Taiwan The survey also showed that it was generally infected with most pigs (Su et al., 2002 Taiwan porcine circovirus infection survey; Taiwan Provincial Animal Husbandry and Veterinary Society and the Republic of China Veterinary Society's 91st annual joint annual conference and academic paper presentation) . PCV2 infection is mainly diagnosed by indirect fluorescent antibody method (IFA) and multiplex PCR (Larocgelle et al., J. Virol. Methods 80(1): 69-75, 1999; Ouardani et al. J. Clin. Microbiol. 37(12): 3917-3924, 1999); however, currently commercially available PCV2 antibodies, and fluorescent antibody test strips, are very expensive. Nawagitul et al. used recombinant baculovirus-infected insect cells to express the Cap recombinant protein as an antigen, and used ELISA to perform large-scale serum tests. The results showed that the sensitivity and specificity were more than 90% (Nawagitul et al., Immunol 9 (1): 33-40, 2002). However, this recombinant protein product requires cell culture and virus proliferation during preparation, which is cumbersome and economical. Pig is an important economic animal in the province and an important animal model for human organ transplantation research. Therefore, it has developed a highly specific and low cost test reagent and immunological binding (eg ELISA) antibody detection kit for screening. It is imperative that pigs infected with PCV2 undergo large-scale serum monitoring.

Thus, the present invention successfully produced recombinant protein fragments encompassing six different regions, A to F, on the Cap protein sequence using the E. coli expression system. Moreover, according to one embodiment of the present invention, after each Cap recombinant fragment is coated on a microplate, the specific pig serum measured by IFA is used for analysis, and it is found that the C-terminal CE fragments of Cap are all good. Antigenicity. All IFA-negative sera were negative, with a specificity of 100%. The positive detection rate of IFA-positive sera was best when C and D were used as antigen fragments, up to 90%. These results show that the in vitro ELISA method established by the specific Cap protein fragment expressed by E. coli has good sensitivity and specificity, and has the potential to be developed into an immunological binding (e.g., ELISA) antibody detection kit. And because it is using E. coli The performance system for antigen manufacturing will effectively reduce the manufacturing cost of detection reagents and can be applied to large-scale routine monitoring of PCV2-infected pig serum.

The objects and advantages of the invention will be set forth in part or in the description.

An object of the present invention is to provide a porcine circovirus capsid protein (Cap) subunit fragment for detecting an anti-porcine circovirus type 2 (PCV2) antibody, which comprises a porcine ring Amino acid sequences of amino acid numbers 101-150, 151-1200, and 101-200 (SEQ ID NOS: 1, 2, and 3, respectively) of the coat protein coat protein.

In a specific aspect of the invention, the viral coat protein subunit fragment is a recombinant protein prepared by an E. coli expression system.

Another object of the present invention relates to a serodiagnosis method for detecting PCV2 infection in pigs using a viral coat protein subunit fragment. According to the present invention, a PCV2 serodiagnostic method having the features of the present invention comprises: contacting a serum sample obtained from a pig to be tested with a viral coat protein subunit fragment of the present invention, and detecting whether there is an antiserum in the serum by immunological binding. -PCV2 antibody.

It is an object of the present invention to provide a diagnostic kit for detecting PCV2 virus infection in pigs, characterized in that it comprises the virus shell egg of the present invention. The white subunit fragment is used as a detection antigen, and an anti-porcine serum secondary antibody and a coloring reagent.

Other features of the invention will become apparent from the following detailed description of the embodiments.

The invention will be described in detail with particular embodiments. These specific examples are provided by way of illustration of the invention and are not intended to limit the invention. The present invention is intended to embrace these and other modifications and variations within the scope and spirit of the invention.

[Examples] Example 1. Recombination of different regions of the viral coat protein to express the plastid structure and its large performance

A plurality of pairs of specific primers (listed in Table 1) were designed for the PCV2 ORF2 gene sequence (GenBank no. AY885255), and nucleic acid fragments corresponding to each region of ORF2 (AF) were amplified by PCR, and sequenced by nucleotides. After confirming the correct sequence, they were separately transferred into the E. coli expression vector (pET32b), and were directed against the PCV2 viral coat protein fragment A (aa 1-50), B (aa 51-100), C (aa 101-150), Six different regions, D (aa 151-200), E (aa 185-233) and F (aa 51-233), were constructed (Fig. 1), and each recombinant plastid was constructed and named as: pET32/Cap1-50. , pET32/Cap51-100, pET32/Cap101-150, pET32/Cap151-200, pET32/Cap185-233 and pET32/Cap51-233.

Then, the reconstructed recombinant plastids were transformed into E. coli BL21 (DE3), and a large number of expressions were performed using the E. coli expression system.

The corresponding oligonucleotide number registered in AY885225 is marked in the scratch number, but the limit The sequence identified by the enzyme is indicated by a box.

The method of inducing a large amount of performance is as follows. The E. coli expression strain containing each recombinant expression plastid was cultured in LB culture medium, and the overnight bacterial solution was taken and re-cultured to LB culture medium at a ratio of 1:50 until the OD ₆₀₀ was 0.6-0.8, and the final concentration was added to 1 mM. The IPTG (isopropylthio-β-D-galactoside) was subjected to a large amount of induction performance for 4 hours, and then centrifuged at 12 000 rpm for 20 minutes to collect the bacterial mass. The pellet was suspended by adding 10 mL of a lysis buffer (100 mM NaH ₂ PO ₄ , 10 mM Tris-HCl, pH 7.0), subjected to ultrasonication to crush the cells, and then centrifuged at 13,000 rpm for 30 minutes. The supernatant is directly mixed with the metal ion resin equilibrated with the dissolution buffer (containing 10 mM imidazole) at low temperature for 16-18 hours, poured into a chromatography column (BIO-RAD), and repeatedly washed through the dissolution buffer several times. The column was washed with a dissolution buffer containing 10 and 20 mM imidazole, and finally the protein was eluted by adding an elution buffer containing 250 mM imidazole. The buffer used in the insoluble protein purification process contained 8 M urea, and finally the urea was removed by dialysis against PBS. The purified expression protein was subjected to protein electrophoresis (SDS-PAGE) and Western blotting analysis using an anti-His tag antibody to determine its antigenicity.

E. coli recombinant expression strains were induced at 37 ° C for 4 hours and analyzed by 12% SDS-PAGE. Obvious performance products were observed and matched to the expected molecular weight (Fig. 2A). Monoantibody against histidine The results of Western blotting analysis also confirmed these recombinant expression proteins (Fig. 2B).

Example 2: Establishing an indirect ELISA antibody detection method using a viral coat protein subunit fragment

The purified Cap recombinant expression proteins were diluted to a concentration of 10 μg/ml in a coating buffer (carbonate buffer, pH 9.6), and 50 μl per well was added to a 96-well EIA microplate (Nunc) for coating at 4 ° C ( Coating) overnight. After aspirating the solution, blocking was performed with 1% BSA/PBS at 37 ° C for 1 hour, and then 50 μl of appropriately diluted (1:200) 6 pig serums which had been determined by IFA were added to each well and allowed to act at 37 ° C. 1 hour. After washing, a properly diluted peroxidase-labeled goat-anti-pig IgG conjugate was added to each well as a secondary antibody and allowed to act at 37 ° C for 45 minutes. After washing, the final addition of the coloring agent was carried out in a dark place at room temperature for a color reaction for 15 minutes, and the absorbance at a wavelength of 405 nm was measured by an ELISA reader (ELISA Reader).

As a result, as shown in Fig. 3, the N-terminal AB fragment of PCV2 Cap did not significantly react in the ELISA (Fig. 3A-B), since the N-terminus of the PCV2 Cap protein may be involved in DNA binding and viral particle assembly. Function, it should not have a major antigenic position. The C-terminal C-F fragments of Cap showed good antigenicity (Fig. 3C-F). The positive and negative serum reaction values were significantly different, and the negative results were clearly determined. Further, the four antigenic fragments were further determined. Determination of ELISA cutoff values. The C-terminal recombinant fragment (CF) and the pET carrier protein of each Cap were separately applied to the EIA microplate (Nunc), and 12 colostrums were provided by Dr. Yang Chengyi from the Taiwan Institute of Animal Science and Technology and negative by IFA. The pig serum was subjected to an indirect ELISA and the judgment value (cutoff value) was further calculated. The results showed that the average OD value for antigen fragment C was 0.106 and the standard deviation (SD) was 0.021; the average OD value for antigen fragment D was 0.122 with a standard deviation of 0.043; the average OD value for antigen fragment E was 0.189 and the standard deviation was The average OD value for antigen fragment F was 0.141 and the standard deviation was 0.049 (see Table 2 below). If the average value plus two standard deviation values (mean +2SD) is used as the criterion, the cut-off values for the CF antigen fragment ELISA analysis are 0.148, 0.208, 0.323, and 0.239, respectively. The standard deviation values (mean + 3 SD) were used as the criterion for the ELISA analysis of the CF antigen fragments, and the cut-off values were 0.169, 0.251, 0.390, and 0.288, respectively.

Twenty-six wild pig serums positive by IFA were randomly selected and subjected to indirect ELISA analysis for C-F antigen fragments. The results of the reaction OD values are shown in Table 3 below. When the average +2SD was used as the cutoff value, the positive detection rates were 96.2% (25/26), 84.6% (22/26), 73.1% (19/26) and 61.5% (16/26), respectively. When the average +3SD is used as the cutoff value, the positive detection rate is 96.2% (25/26), 84.6% (22/26), 69.2 (18/26) and 61.5% (16/26).

Therefore, the above results show that the ELISA antibody detection method established by the two antigen fragments C and D of the PCV2 Cap recombinant protein prepared according to the invention has good sensitivity and specificity and can be further developed into use. An immunological assay diagnostic kit for detecting PCV2 infection in pigs.

Example 3: Viral coat protein CD subunit fragment (amino acid number 101-200) Recombination performance and construction of the body

From the results of the above Example 2, the porcine circovirus coat protein (Cap) C and D antigen fragments have good sensitivity and specificity, so we further designed to construct a CD fragment recombinant protein expression vector. First, the CD nucleic acid fragment of Cap was amplified by PCR, and then transferred into an E. coli expression vector (pET32a), thereby constructing a CD fragment recombinant expression vector (pET32aCap101-200). The recombinant vector was transformed into E. coli BL21 (DE3), and the expression of the recombinant protein was performed after the sequencing analysis was correct.

After the recombinant Escherichia coli recombinant strain was induced to express a large amount of recombinant protein by 1 mM IPTG, the pellet obtained by centrifuging the bacterial solution was dissolved in a native lysis buffer, followed by ultrasonication and centrifugation, and the precipitate was partially used. The denatured lysis buffer was reconstituted, and the recombinant protein was purified by ion affinity column chromatography and dialyzed against PBS, and then confirmed by protein electrophoresis (SDS-PAGE) and Western blot analysis.

After 4 hours of IPTG induction at 37 °C, the E. coli recombinant expression strain was analyzed by Western blotting with 15% SDS-PAGE and anti-histamine single-source antibody. Obvious performance products were observed and The expected molecular weight of 30 kDa is consistent, while most recombinant proteins are insoluble. Further restructuring The protein was purified by ion affinity column chromatography and analyzed by 15% SDS-PAGE after dialysis, showing that the CD recombinant protein was efficiently purified (see Figure 4).

Example 4: Indirect ELISA antibody detection method based on viral coat protein CD subunit fragment

The assay of CD recombinant protein fragments was performed according to the same indirect ELISA method as described in Example 2. The results of the reaction of 12 IFA-negative pig sera showed an average OD value of 0.182 for the antigen fragment CD and a standard deviation (SD) of 0.061 (results are shown in Table 4). If the mean plus two standard deviations (mean +2SD) is used as the criterion, the cut-off value of the CD antigen fragment is 0.001, and the cutoff value is 0.304; +3SD) As a criterion, the cutoff value is 0.365 (as shown in Table 4 below). Analysis of the response values of indirect ELISA analysis using the same 25 IFA-positive field pig sera showed that the positive detection rate was 92% (23/25) with an average of +2SD as the cut-off value, and the average +3SD. When the cutoff value is used, the positive detection rate is 88% (22/25).

No colostrum was obtained and was negative for pig serum by IFA.

The results showed that the CD fragments constructed and expressed longer were tested by indirect ELISA and the sensitivity was 92%. Since the Cap protein sequence of PCV2 is known to be highly reserved in each isolate (Kamstrup et al, Vaccine 22, 1358-1361, 2004), recombinant protein C, D and CD subunit antigens of PCV2 Cap according to the present invention are known. The ELISA antibody detection method established by the fragment can have good sensitivity and specificity for the anti-PCV2 antiserum of pigs, so it can be further developed into a diagnostic reagent kit for immunological binding analysis for detecting PCV2 infection in pigs. Further, mass production by an Escherichia coli host can significantly reduce the antigen cost compared with the conventional diagnostic reagent, and therefore has both industrial applicability and advancement.

Figure 1. Schematic representation of each Cap subunit (A-F) fragment located in the coding region of PCV2 ORF2. Wherein fragment A corresponds to amino acid number 1-50, fragment B corresponds to amino acid number 51-100, fragment C corresponds to amino acid number 101-150, fragment D corresponds to amino acid number 151-200, and fragment E corresponds to amino acid number 185-233 And fragment F corresponds to amino acid number 51-233.

Figure 2. Performance of Cap subunit A-F in E. coli host, analyzed by SDS-PAGE (A) and Western blotting (B) using anti-His antibody. Lines 1-6 are the Cap unit fragments A-F that are represented. The position of each expressed recombinant protein is indicated by an arrow.

Figure 3. Results of an indirect ELISA reaction of pig serum against each Cap unit fragment. Pig serum samples included two negative by IFA (IFA-) and four positive by IFA (IFA+). Figures (A) to (F) are optical density values (OD values at 405 nm) for the reaction of Cap subunit A-F fragments, respectively.

Figure 4. Cap subunit fragment CD, which was purified by ion affinity column chromatography and analyzed by 15% SDS-PAGE. Figure 1 (A) of the first act of bacterial cell lysate, the second act of washing the column through the liquid, the 4-6 behavior of the CD subunit fragment rushed through the column with the dissolution buffer containing 250 mM imidazole . Figure (B) Results of the first CD fragment of the purified CD subunit fragment after dialysis by PBS.

<110> National Chung Hsing University

<120> Porcine circovirus type 2 coat protein subunit fragment and use thereof

<160> 3

<170> Patent In Version 3.3

<210> 1

<211> 50

<212> PRT

<213> Porcine circovirus type 2

<400> 1

<210> 2

<211> 50

<212> PRT

<213> Porcine circovirus type 2

<400> 2

<210> 3

<211> 100

<212> PRT

<213> Porcine circovirus type 2

<400> 3

Claims (12)

A porcine circovirus capsid protein (Cap) subunit fragment for detecting an anti-porcine circovirus type 2 (PCV2) antibody, characterized by an amino acid sequence selected from a porcine circovirus coat protein The amino acid sequence consisting of 101-150, 151-1200, and 101-200 (SEQ ID NOS: 1, 2, and 3, respectively), and is a recombinant protein prepared by the E. coli expression system. A serodiagnosis method for detecting PCV2 infection in pigs, which utilizes a viral coat protein subunit fragment according to claim 1 of the scope of the patent application, the serodiagnostic method comprising: a serum sample obtained from a pig to be tested and based on The viral coat protein subunit fragment of the first application of the patent scope is contacted, and the anti-PCV2 antibody is detected in the serum by immunological binding. The serodiagnosis method according to claim 2, wherein the immunological binding method is an indirect ELISA assay. The serodiagnosis method according to the second aspect of the patent application, wherein the immunological binding method is an indirect fluorescent antibody method (IFA). The serodiagnosis method according to the first aspect of the invention, wherein the viral coat protein subunit fragment consists of the amino acid sequence of amino acid number 101-150 (SEQ ID NO: 1) of the Cap recombinant protein. The serodiagnosis method according to the first aspect of the invention, wherein the viral coat protein subunit fragment consists of the amino acid sequence of the amino acid number 151-1200 (SEQ ID NO: 2) of the Cap recombinant protein. The serodiagnosis method according to the first aspect of the invention, wherein the viral coat protein subunit fragment consists of the amino acid sequence of amino acid number 101-200 (SEQ ID NO: 3) of the Cap recombinant protein. A diagnostic kit for detecting porcine circovirus type 2 virus (PCV2) infection in pigs, characterized by comprising a viral coat protein subunit fragment according to claim 1 of the patent application scope as a detection antigen, and anti- Porcine serum secondary antibody and coloring reagent. The serodiagnosis method according to the eighth aspect of the patent application, wherein the viral coat protein subunit fragment is composed of the amino acid sequence of the amino acid sequence 101-150 (SEQ ID NO: 1) of the Cap protein. to make. The diagnostic kit according to item 8 of the patent application, wherein the viral coat protein subunit fragment consists of the amino acid sequence of the Cap protein amino acid number 151-1200 (SEQ ID NO: 2). The diagnostic kit according to item 8 of the patent application, wherein the viral coat protein subunit fragment consists of the amino acid sequence of the amino acid sequence 101-200 (SEQ ID NO: 3) of the Cap protein. The diagnostic kit according to item 8 of the patent application, wherein the secondary antibody is an oxidase-labeled anti-porcine IgG antibody.