KR20190050961A - Method of Extract Solution for Detecting PWN Infection using the EXPB3-Mab-3-1-H3-C11-E4(EXPB3-C11-E4) - Google Patents

Abstract

The present invention relates to a method for preparing an extract for a PWN-infection test using EXPB3-Mab-3-1-H3-C11-E4(EXPB3-C11-E4), which comprises: a step (1) of collecting a wood pellet from a suspected PWN-infection pine tree; and a step (2) of extracting the wood pellet collected in the step (1) with a PBS extract. The extract prepared by the method of the present invention can perform a rapid and accurate PWN-infection test.

Description

[0001] The present invention relates to a method for producing a PWN-infectious test extract used for a PWN-infection test using EXPB3-Mab-3-1-H3-C11-E4 (EXPB3- EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4)}

The present invention relates to a method for producing PWN-infectious test extract used for PWN-infection test using EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4).

Pine wilt disease (PWD) is currently the most destructive coniferous forest disease in the world and has been found to be due to infection with pine nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhner, 1934) and Nickle (Futai, 2013). The disease was first reported in Nagasaki, Japan in 1905, and spread to other East Asian countries, including China in 1982, Taiwan in 1985, and Korea in 1988 (Futai, 2013; Suzuki, 2002). In addition, pine trees infected with PWN have been observed in the Iberian Peninsula of Portugal in 1999 (Futai, 2013; Mota et al., 1999) and were observed in 2008 in the Caceres region of Spain near Portugal (Abelleira et al., 2011). The proliferation of PWN in the area is due to Monochamus spp. It is mediated by several pine sawdust beetles species. However, it is known that outbreaks of PWD occur in remote areas or on other continents due to the artificial transport of PWN and its infected trees (Futai, 2013).

Therefore, the best way to prevent current widespread spread of PWD is to prevent the transfer of pine trees from PWN-infected areas to other uninfected areas. Finding and burning PWN-infected trees is one of the most effective ways to prevent the spread of PWD (Futai, 2013), because removal of infected trees simultaneously reduces the density of the medium.

A variety of research efforts have been conducted to identify and detect PWNs and infected pines. Identification of PWN-infection in pine trees was based on microscopic observation of nematodes from pine trees (Futai, 2013; Yik and Birchfield, 1981).

(Cheng et al., 2008; Kanzaki and Futai, 2002) and various microsatellites (Jung et al., 2010), including mitochondrial genes (Cheng et al., 2008; Mallez et al., 2013) was identified and used for technology development.

However, most of the molecular tools require a sophisticated machine and sometimes the PCR should be separated from the agarose gel to complete the diagnosis. To overcome these limitations, several methods of using ring-mediated isothermal amplification (LAMP) have been developed (Kang et al., 2015; Kikuchi et al., 2009). However, the LAMP reaction mixture should be incubated at about 60 ° C for at least 1 hour and a strong fluorescence signal should be identified by UV light to extract the genomic DNA from the wood phosphors.

To overcome these limitations, it is required to develop kits that can perform rapid tests using antigen-antibody interactions for rapid diagnosis in the field.

Korean Patent Publication No. 10-2010-0045817

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems of the prior art,

EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4) which is a monoclonal antibody for PWN-infection test which makes it possible to perform PWN-infection test quickly and accurately by antigen- , A method for producing a monoclonal antibody for PWN infection test, a method for testing PWN infection using EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4) It is another object of the present invention to provide a method for producing an extract for PWN-infection test which can improve the accuracy of the infection test.

The present invention

(a) blending the amino acid sequence of EXPB3 with the Bursaphelenchus xylophilus gene database to design a peptide that exhibits amino acid sequence mismatch for PWN and EXP homologues of other nematode species;

(b) binding the peptide designed in step (a) with keyhole limpet hemocyanin (KLH) and then immunizing Balb / C rats using the peptide as an antigen to obtain a fusion hybridoma;

(c) primary screening the supernatant of the fusion hybridomas obtained in step (b) by ELISA using the antigen prepared in step (b);

(d) collecting wood pellets from PWN-infected pine trees (PWNIPTs) and extracting with PBS extract;

(e) Hybridoma strain first screened in step (c) is secondly screened by ELISA using the PBS extract obtained in step (d) as an antigen, and OD (optical density) Selecting a clone by reference; And

(f) performing a limited dilution on the clone selected in step (e), and then screening the monoclonal antibody by ELISA using the PBS extract obtained in step (d) as an antigen;

A method for producing a monoclonal antibody for detecting PWN-infected pines comprising the steps of:

The present invention also relates to

(1) collecting wood pellets from PWN-suspect pine trees; And

(2) extracting the wood pellets collected in the step (1) with PBS extract;

A method for preparing an extract for PWN-infectivity test.

The present invention also relates to

EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4) which is a monoclonal antibody for PWN-infection test.

The present invention also relates to

(A) collecting wood pellets from PWN-suspect pine trees; And

(B) extracting the wood pellets collected in the step (A) with a PBS extract; And

(C) Performing ELISA using EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4) which is a monoclonal antibody for PWN-infection test and PBS extract in step (B) ;

The method comprising the steps of:

EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4), a monoclonal antibody for the PWN-infection test of the present invention, can be used to rapidly and accurately test for PWN infection by antigen- Can be performed.

In addition, it can be very usefully used in the production of a diagnostic kit capable of conducting the PWN-infection test as described above.

In addition, the extract prepared by the method of the present invention for producing an extract for PWN-infectious test enables rapid and accurate testing of PWN-infection.

Figure 1 is a graph showing the immunoreactivity of each extract of EXPB3-Mabs.
Figure 2 is a graph showing the immunological response of 96 selected hybridomas to the PBS extract of PWNIPT.
Figure 3 shows the results of limited dilution of two EXPB3 hybridomas clones.
Figure 4 shows the results of measuring the amount of antigen present in each of 74 PWNIPTs collected at 9 different sites using EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4) .
5 is a graph showing the results of primary screening using the supernatant of a fusion hybridoma cell line for two EXPB3 peptides.
FIGS. 6 to 8 are graphs showing limiting dilution results of nine EXPB3 hybridomas clones showing low normalized specificity. FIG.

The present invention

(a) blending the amino acid sequence of EXPB3 with the Bursaphelenchus xylophilus gene database to design a peptide that exhibits amino acid sequence mismatch for PWN and EXP homologues of other nematode species;

(b) binding the peptide designed in step (a) with keyhole limpet hemocyanin (KLH) and then immunizing Balb / C rats using the peptide as an antigen to obtain a fusion hybridoma;

(c) primary screening the supernatant of the fusion hybridomas obtained in step (b) by ELISA using the antigen prepared in step (b);

(d) collecting wood pellets from PWN-infected pine trees (PWNIPTs) and extracting with PBS extract;

(e) Hybridoma strain first screened in step (c) is secondly screened by ELISA using the PBS extract obtained in step (d) as an antigen, and OD (optical density) Selecting a clone by reference; And

(f) performing a limited dilution on the clone selected in step (e), and then screening the monoclonal antibody by ELISA using the PBS extract obtained in step (d) as an antigen;

To a method for producing a monoclonal antibody for detecting PWN-infected pines.

In this study, Mabs selectively recognizing PWN-infected pine (PWNIPTs) were constructed by constructing a monoclonal antibody (Mab) that secretes a fusion hybrid library for EXPB3. In addition, the specificity and sensitivity of Mab were confirmed using various PWNIPTs collected in 9 Korean regions

In the method for producing the monoclonal antibody, the sequence of the peptide obtained by the peptide design in the step (a) may be 42-CGLDIANLSAGVSSKLFD-59 and 141-KPATITYLKCSDTTPTTNC-150.

Collection of wood pellets from PWN-infected pine trees (PWNIPTs) in step (d) is preferably carried out in two or more pine trees. Specifically, in the present invention, as shown in Tables 1 to 3, 74 PWNIPTs and healthy pine trees (HPT) 22 using a 9-region wood pellet collector (FJ Technology Co., Ltd., Seoul, Korea) Wood pellets were collected from dogs and used.

In the method for producing a monoclonal antibody of the present invention,

The extraction in step (d) may be performed by mixing the PBS solution with the wood pellet and incubating at room temperature. The PBS solution and the wood pellet are mixed at a weight ratio of 1: 0.5 to 0.5: 1, and the incubation time Is preferably 3 to 20 minutes.

The present invention also relates to

(1) collecting wood pellets from PWN-suspect pine trees; And

(2) extracting the wood pellets collected in the step (1) with PBS extract;

And a method for producing the extract for PWN-infection test.

The extraction in the step (2) is preferably carried out by mixing the PBS solution with the wood pellet and incubating at room temperature.

The PBS solution and the wood pellets are mixed at a weight ratio of 1: 0.5 to 0.5: 1, and the incubation time is preferably 3 to 20 minutes.

The present invention also relates to

EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4) which is a monoclonal antibody for PWN-infection test.

EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4) can be prepared by the method of the present invention.

The present invention also relates to

(A) collecting wood pellets from PWN-suspect pine trees; And

(B) extracting the wood pellets collected in the step (A) with a PBS extract; And

(C) An ELISA was carried out using the EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4) as the monoclonal antibody for PWN-infection test of paragraph 9 and the PBS extract of step (B) ;

And a method for testing PWN infection.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are intended to further illustrate the present invention, and the scope of the present invention is not limited by the following examples. The following examples can be appropriately modified and changed by those skilled in the art within the scope of the present invention.

1. Example

(One) Identification and synthesis of target proteins of peptides against antigens

In a previous study, the present inventors reported that ExpansinB3 (EXPB3) was highly expressed in the propagation and dispersion steps of PWN (Lee et al., 2012). The amino acid sequence of EXPB3 can be found in the B. xylophilus database of GeneDB (http://www.genedb.org/Homepage/Bxylophilus) and NCBI Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) Used to blast. Based on the Blast results, two peptides were designed that exhibit amino acid sequence mismatches for various EXP homologues of PWN and other nematode species. The sequences of the two peptides synthesized were 42-CGLDIANLSAGVSSKLFD-59 and 141-KPATITYLKCSDTTPTTNC-150. Two peptides were conjugated with keyhole limpet hemocyanin (KLH) and then used as an antigen to immunize four Balb / C mice.

(2) EXPB3-specific monoclonal antibody immunization and primary screening

Fusion hybrid hybridoma cell lines constituting the experiment were approved by the Laboratory Animal Ethics Committee of Hallym University (No. HMC2016-00622-7). The present inventors followed the standard monoclonal antibody production protocol used in Lee et al. (Lee et al., 2011). The supernatants of the fusion hybridomas recognizing the two peptide antigens were screened using an enzyme linked immunosorbent assay (ELISA). The 96-well plate was coated with the antigen peptide (0.1 μg / ml) and then blocked by shaking for 1 hour at room temperature (RT) with 5.0% nonfat dry milk in PBS. After washing three times with 0.1% Triton X-100 in PBS (PBST), the supernatant of the 1920 fusion hybridoma cell line was added and incubated overnight at 4 ° C. After washing three times with PBST, peroxidase conjugated anti-mouse IgG, A and M antibodies (ThermoFisher Scientific, Waltham, MA, USA) were applied and incubated at room temperature for 2 hours. Step 1 - Peroxidase activity was detected using Ultra TMB solution (ThermoFisher Scientific) and measured using Epoch ELISA plate reader (Biotek, Winooski, VT, USA).

(3) Manufacture of wood pellets and extracts

Wood pellets were collected from 74 PWNIPT and 22 healthy pine trees (HPT) using a wood pellet collector (FJ Technology Co., LTD, Seoul, Korea) in 9 regions or 3 regions of Korea (Table 1) . The collected wood pellets were immediately transferred to the laboratory and refrigerated at 4 ° C until use.

To determine the optimal extraction solution, we used dH ₂ O alone or in combination with 0.1% Triton X-100 (H ₂ OT). And either phosphate buffered saline (PBS, 0.01 M sodium phosphate, 0.154 M NaCl, pH 7.2) was used alone or in combination with 0.1% Triton X-100 (PBST). A total of 5 ml of the four extraction solutions was mixed with 0.5 g of wood pellets and incubated at room temperature for 5 minutes and then the solution was poured carefully to avoid debris. The PBS extract in solution showed a higher immune response to other selected supernatants (Figure 1). Therefore, in this study, all other experiments were performed using PBS as an extract.

As shown in Fig. 1, EXPB3-Mabs showed relatively high immunoreactivity with PBS extract. Compared to PBS with H ₂ O or 1.0% Triton X-100 (PBST) extracts containing H ₂ O, 1.0% Triton X-100 (H ₂ OT), PBS extracts showed high optical density . The extracts of the two PWNIPT extracts from Jeju Island, Songsan and Iksan, Jeollabukdo showed higher OD values than the other extracts. However, the OD value of H ₂ O extracts extracted from PWNIPT of Gyeongnam Province was higher.

(4) Second screening of 74 PWN-infected pines with PBS extract

The 96 hybrid hybrid lines selected in (1) above were subjected to secondary screening in the same manner, except that 74 PWNIPT PBS extract was used as an immunogen. This screening was performed in three replicates. Twelve clones with high OD were selected for single cell replication.

(5) Single cell replication by limiting dilution

As mentioned earlier, a limited dilution was performed to obtain a single cell clone out of 12 hybridoma strains showing higher OD in the mixed PWNIPT (Lee et al., 2011). The supernatant from the limited dilution clones was screened using mixed PBS extracts of 74 PWNIPT. Twenty - two healthy pine tree (HPT) PBS extracts from three Korean regions were collected and used as a control. The OD of each Mab for the mixed PBS extract of 74 PWNIPTs was normalized to the mixed PBS extract of 22 HPTs by the OD of the same Mab. The equation is as follows:

Normalized specificity = (OD of OD-22 HPT mixed PBS extract of 74 PWNIPT OD of PBS extract / 22 OD of mixed PBS extract of HPT)

(6) EXPB3-Mab-3-1-H3-C11-74 Standardized specificity measurement of PWNIPT

EXPB3-Mab-3-1-H3-C11 had limited dilution and EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4) had the strongest reactivity to mixed PBS extract of PWNIPT (Fig. 3). Because there may be unknown variations between PWNIPTs depending on area and stage of infection, we used 74 PWNIPT PBS extracts from nine Korean regions. Hybridoma cell culture media were used as control and background signals. The selected hybridoma lines were further screened using 74 PWNIPTs. In this study, 42 red pine trees and 32 black pine wood pellets were used (Tables 1-3). The range of progression of PWD in the pine trees used in the study is distributed from normal (0) to complete disappearance (6) by visual inspection and presence of PWN. In addition, the perimeter of infected trees was measured at 1.2 m above the ground and used for correlation analysis (Tables 1-3).

(7) Statistical analysis

Differences between groups were analyzed using an Excel program (Microsoft, Redmond, WA, USA) ANOVA. Tukey-Kramer post-mortem analysis was performed using an Excel program (Microsoft) to identify differences between groups. Optical density, disease progression and tree perimeter Pearson correlation coefficients at 1.2 m above ground were calculated using the Excel program (Microsoft).

2. Experimental results

(1) 96 hybrid hybridomas secreting EXPB3 antibody were identified by primary screening using antigenic peptides.

The EXPB3 monoclonal antibody (Mab) library containing 1,920 fused hybridoma lines was generated for Mab screening to recognize PWNIPT. The primary screening was performed by ELISA to find a fusion hybridoma line that secretes the EXPB3 peptide recognizing the Mab used as an antigen. Because there is a slight difference between the plates, the OD of each 96 plate was standardized using the highest OD on each plate. The cut-off value of the normalized OD obtained in the ELISA was at least 40% of the highest OD of each plate. After primary screening, 96 fusion cells were selected for further study (Figure 1).

(2) PBS was a solution suitable for extracting EXPB3-Mabs antigen

Choosing a solution that can extract the most efficient antigen is one of the important steps in the development of diagnostic technology. It is especially important to select extraction solutions that do not contain health or environmentally hazardous chemicals. This is because diagnostic methods used in the field can be a problem if the solution contains substances harmful to humans or the environment. Therefore, we selected dH ₂ O and PBS with or without 0.1% Triton X-100. LD50 of Triton X-100 is 1,800 mg / kg orally for rats (MSDS, 2013). Therefore, the addition of 0.1% Triton X-100 to the solution does not cause serious toxicity to humans and other wildlife.

Four extracts were subjected to ELISA using four EXPB3-Mab3-1-H3-C11 subclones to compare extraction efficiencies of four solvents (Fig. 1). EXPB3-Mab-3-1-H3-C11-E3 and -E4 of the four EXPB3-Mabs had a higher OD compared to the two other Mabs. The OD showed a similar OD trend to the four extract solutions and the three PWNIPTs, although there was a difference between the four Mabs. Compared with H ₂ O, H ₂ OT and PBST extracts, PBS extracts from PWNIPT of Jeju, Pyeoson and Iksan, Jeollabukdo showed relatively high OD. However, dH ₂ O extracted from PWNIPT of Gyeongnam Nuclear Plant showed OD higher than other extracts (Fig. 1). These results suggest that PBS is suitable for extracting antigen from PWNIPT.

(3) Nine hybridoma strains showed specificity for PBS extract of PWN-infected pine.

The specificity of each hybridoma was analyzed by ELISA to identify the hybridoma lines that secrete PWNIPT - recognizable Mabs among 96 selected hybridoma lines using PWNIPT 's PBS extract. 74 PWNIPTs, including 42 red pines and 32 black pines, were obtained in 9 PWN regions (Tables 1-3 and 4). PBS extracts were collected from 74 PWNITs and used for secondary screening. ODs of 96 hybridoma lines were standardized using EXPB3-Mab-3-3-D9, which has the highest OD. Nine hybridoma lines showed normalized OD values of greater than 0.2 (Figure 2).

(4) The single clones of EXPB3-Mab-3-3-D9 and -3-3-H3 have a high standardization specificity for PWNIPT.

To establish a single cell clone, 13 lines of cells, including 9 lines with an OD value of 0.2 or greater, showed limited dilution in 96 well plates. 96 wells of supernatant were screened using 74 PWNIPT and 22 HPT synthetic PBS extracts to calculate standardized specificity. Of the thirteen strains examined, two strains of EXPB3-Mab-3-1-C9 and -3-2-G11 strains failed and ELISA could not be performed. Five hybridoma lines containing EXPB3-Mab-3-2-E1, EXPB3-Mab3-2-G9, EXPB3-Mab-3-2-F1, EXPB3-Mab-3-2-E1 and EXPB3- Of the single cell clone -3-1-G4 showed very low standardization specificity (< 1.0) (Fig. 7, E). Single cell clones from EXPB3-Mab-3-1-C3, EXPB3-Mab-3-1-F4, EXPB3-Mab-3-1-A5 and EXPB3- (AD of Figures 6 and 7). A single clone of two lines of EXPB3-Mab-3-3-D9 (EXPB3-D9) and EXPB3-Mab-3-1-H3 (EXPB1-H3) showed a higher standardization specificity than the other line of single clone ). Three of the single cell clones of EXPB3-D9 and EXPB1-H3 had a normalized specificity of 3.4 or greater. The clone was used in subsequent studies.

Figure 3 shows the results of limited dilution of two EXPB3 hybridomas clones. That is, to select a single clone from 13 EXPB3 hybridoma lines, a limited dilution was performed (A). EXPB3 - Mab - 3-1 - normalized specificity of supernatant in limiting dilution cells in D9, (B) normalized specificity of 48 supernatants in limiting dilution cells in EXPB3 - Mab - 3-1 - H3. Two clones of each strain were selected for further analysis.

(5) Variations of PWNIPT revealed by EXPB3 antibody

The degree of disease progression of PWNIPT used in the study of the present invention was determined only by the external properties of the tree upon pellet harvesting. Therefore, there is no information about the detailed infection period or physiological change of each pine tree. We measured the amount of antigen present in each of the 74 PWNIPTs collected at 9 different sites using the developed EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4) 4A).

The OD value of EXPB3-C11-E4 of red pine was compared with the OD value of black pine, and the OD value of black pine was found to be statistically higher than that of red pine (Fig. 4B). Black pines collected from three regions of Cheju Island tended to have OD higher than those of the mainland when comparing 32 black pines collected in five regions of Korea (Fig. 4C). However, there were no statistically significant differences in the 42 red pines collected from seven regions of Korea (Fig. 4D).

There was no correlation between the OD of the red pines and disease progression (Pearson correlation coefficient = -0.174). However, there was a low correlation between tree circumference (1.2 m above the ground) and OD (1.2 m above the ground) and disease progression (Pearson correlation coefficients: - 0.21529 and 0.34562). In contrast, there was a high correlation between OD and OD and disease progression at 1.2 m above ground (Pearson correlation coefficient: 0.49583 and 0.43023). However, the correlation between the degree of disease progression and the tree circumference at 1.2 m (Pearson correlation coefficient: -0.21069) on the black pines was low. The results of this analysis suggest that there is a difference in EXBP3-Mab reactivity depending on species, distribution area and stage of development of individual black pine trees.

3. Results Discussion

The main goal of this study is to establish a method for collecting wood pellets to generate an array of EXPB3-Mabs that can be used for rapid diagnosis of PWNIPT in situ and to complete an accurate diagnosis. To achieve this goal, we first identified a suitable solution to extract the antigen most efficiently from the PWNIPT pellet. A PBS extract of PWNIPT was used to identify the most specific MAB. EXPB3-Mabs were screened to increase the accuracy and sensitivity of EXPB3-Mabs using high reactivity to PBS extracts of PWNIPT as well as low reactivity to PBS extracts of HPBs. Thus, EXPB3-Mabs was selected based on the normalized sensitivity calculated by a methodology with high specificity and sensitivity to PWNIPT. PWNIPT has been collected from trunk wood pellets or pellets of 1.3 m in height since the publication of another research report on PWN detection in PWNIPT (Magnusson et al., 2007; Schroder et al., 2009). The present inventors have set the sampling position to be 1.2 m above the ground in consideration of the physical characteristics of Korean adults. This methodology has been applied similarly to all samples to increase applicability in the field.

According to the study of the present invention, black pine trees showed significantly higher reactivity to EXPB3-Mabs than red pine trees (Fig. 4B). In addition, the reactivity to black pine trees showed a significant correlation with the degree of disease progression and the perimeter of trees. This result suggests that PWD progression in black pine can be predicted by reactivity to EXPB3-C11-E4 antibody, unlike red pine.

EXP is an enzyme present in plants and plays an important role in mediating cell wall relaxation and allows for normal development by reducing adhesion between adjacent cell wall polysaccharides (Marowa et al., 2016). In addition, EXPs of plant parasitic nematodes have similar functions to those of plant EXPs and thus can help to invade nematodes in host plants (Qin et al., 2004). The EXP3-Mabs developed in this study are expected to be very valuable data for studying the invasion and breeding mechanism of PWN in pine trees as well as the development of rapid detection tools.

Claims (3)

(1) collecting wood pellets from PWN-suspect pine trees; And
(2) extracting the wood pellets collected in the step (1) with PBS extract;
&Lt; RTI ID = 0.0 &gt; PWN-infection &lt; / RTI &gt; The method according to claim 1,
Wherein the extraction in step (2) is performed by mixing the PBS solution with the wood pellet and incubating at room temperature. 3. The method of claim 2,
Wherein the PBS solution and the wood pellets are mixed at a weight ratio of 1: 0.5 to 0.5: 1, and the incubation time is 3 to 20 minutes.

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