KR20080109687A - Purification method of infectious virus from blood - Google Patents

Abstract

A purity separation and refining method of the virus from the infectious blood is provided to easily separate and refine the virus from the blood of human or animal, thereby facilitating the development of infectious virus separating and refining kit, and production of antibody. The virus, porcine circovirus type 2(PCV2) is separated and refined from the infectious blood of human or animal through the filtration by using celite, clarification of host material and impurities by using organic solvent, virus concentration by using polyethylene glycol(PEG) as water-soluble precipitation supplement, and the virus fractionation by using sucrose density gradient centrifugation.

Description

Purification method of infectious virus from blood

The present invention provides a process for easily separating and purifying a virus from human or animal blood for the purpose of developing an infectious virus isolation purification kit and producing an antibody.

Clinical samples for virus screening are collected from patients or hosts suspected of viral infection. Typical clinical samples are blood, urine and feces, pharyngeal fluid, organs or tissues. If a small amount of virus is present in the sample and it is not easy to detect, the cells or cells of known characteristics derived from human or animal tissues are cultured in a sterile medium and then inoculated with the sample containing the virus to infuse a large amount of the virus. Specific viruses can then be purified and purified. Cell culture is first pulverized tissues cut from humans or animals and then separated into individual three seconds by proteolytic enzyme treatment and put them in a special surface-treated plastic container with sterilized culture medium and cultured at a constant temperature. If possible, use an incubator with 5-10% CO ₂ to minimize pH changes.

Cells proliferated by the method specified above adhere to the surface of the container and spread flatly to form a monolayer. The culture is composed of buffers containing essential nutrients such as sugars, salts, vitamins and amino acids in concentrations similar to those of living organisms (pH 7.2-7.4). In addition, antibiotics to prevent animal serum and bacterial contamination required for cell growth may be added to the culture. Virus detection by cultured cells has the advantage of observing the cytopathic effect (CPE) characteristic of host cells, measuring the presence of infection, and purely separating specific viruses as the virus proliferates. And contamination are pointed out as disadvantages.

Viruses that circumvent the local defenses of animals or humans frequently spread throughout the blood throughout the body. That is, viremia occurs in which mature virus particles move freely in blood vessels or through infected lymphocytes, and the concentration of the virus is increased through primary and secondary viremia. Therefore, when a large amount of virus is present in a sample such as blood, a method of directly separating and purifying the virus from blood without using cultured cells may be an effective alternative to reduce time and cost. However, since humans can be rapidly infected by accidentally infected blood or blood products, such as viruses that cause hepatitis or AIDS, there is an absolute need for a method of easily and purely separating pathogenic viruses from blood.

In order to achieve the above object, the present invention attempted to isolate and purify porcine circovirus type 2 (PCV2) infected with porcine blood as a model to rule out the potential risk of viruses that can infect humans. For this purpose, filtration using celite, clarification for removal of host substances and impurities using organic solvents, concentration of virus using polyethylene glycol 8000 (PEG), an aqueous precipitation aid, and sucrose density Virus fractionation using gradient centrifugation, and virus concentration process using 10% sugar cushion centrifugation were performed. Hereinafter, the solution of the present invention will be described in more detail.

Cellular proteins, DNA and other substances present in abundance in the blood sometimes interfere with the search for viruses with DNA as a genetic material. In particular, the host nuclear and mitochondria DNA concentrations overwhelm the concentration of viral DNA, which is a major factor preventing the amplification of viral DNA fragments using PCR in blood samples. Therefore, a filtration process for selectively removing impurities from blood and the treatment of organic solvents are required to effectively remove cellular proteins. Organic solvents such as chloroform and n-butanol used in the present invention not only denature cell proteins but also destroy the mitochondrial membrane to form a state in which mitochondrial DNA can be dissolved in DNAase (DNase I). On the other hand, even if the DNAase is treated, the DNA of the virus can be protected by a capsid protein.

In order to collect the virus suspended in the blood after removing the impurities in the above manner, the ultracentrifugation of a certain amount of serum at a speed of 100,000 g or 40,000 rpm causes the particles of the virus to settle on the bottom of the tube. Therefore, the supernatant can be removed and a buffer can be added to the pellet to obtain a virus suspension. The easiest way to do this is to settle together the micro host material present in the blood, and the more serious problem is that the virus can be precipitated by high speed and gravity, resulting in particle damage and loss of infectivity.

In order to solve the above problems, the present invention precipitated the virus by centrifugation at low speed using PEG as an aqueous precipitation aid. Alternatively, the sugar solution was dissolved in a buffer solution to form a cushion at the bottom of the tube so that the virus precipitated by ultra-centrifugation was suspended in the high-density sugar solution layer to protect the virus particles from damage.

Since the virus sample obtained by the centrifugation method is still contaminated with cell material, the sugar solution density gradient centrifugation method was performed for the purpose of purifying more purely to fractionate the sugar solution layer corresponding to the virus size to obtain the virus. All.

The present invention provides a process for easily separating and purifying viruses from human or animal blood, which can be useful for developing infectious virus isolation and purification kits and producing antibodies. Hereinafter, the specific method of the present invention will be described in detail with reference to Examples. However, the scope of the present invention is not limited to the following examples.

{ Example }

1. Pig Blood

Porcine blood used in this study was named as follows after being sold at slaughterhouses in Nonsan (3 points) and Okcheon (7 points) in Chungbuk, Korea (PCU-1, -2, -3, -4, -5,-). 6, -7, -8, -9, -10).

2. PCR analysis

(1) PCV2 capsid protein gene specific primer

To confirm PCV2 infection, DNA was isolated from the porcine blood using Viral DNA / RNA Extraction Kit (iNtRon Biotechnology, Korea). For PCR amplification, specific nucleotide sequences of the following PCV2 envelope protein genes were used as primers.

Forward primer: 5'-CCATGGAAATGACGTATCCAAGGAGG-3 '

Reverse primer: 5'-AAAATCCATGGGCATCTTCAACACCCGC-3 '

(2) PCR reaction

First, PCR was performed using DNA extracted from three blood samples distributed in Okcheon. 50 μl of the reaction solution is 10 × buffer (iNtRon Biotechnology Inc., Korea), 10 ng DNA, 2,5 mM dNTPs, 50 pmol forward and backward primers, 1.5-3.5 mM MgCl ₂ , 1U r Taq It consisted of polymerase. PCR reaction was followed by 1 cycle of denaturation of DNA at 95 ° C for 2 minutes, followed by 35 cycles of continuous reaction (denaturation: 1 minute at 94 ° C, annealing: 2 minutes at 61 ° C, amplification: 3 minutes at 72 ° C). The final treatment was at 5 minutes for 72 minutes.

Experimental results, MgCl ₂ No target band was detected at 1.5 mM, but PCV2 envelope protein gene 702 bp was synthesized at 3.5 mM concentration (FIG. 1). Therefore, it was confirmed that PCV2 was infected in the blood sample.

Secondly, PCR was performed using a commercial PCV2 detection kit (VeTek ™ PCV2 detection kit, iNtRon Biotechnology Inc., Korea) as a template of DNA extracted from seven blood samples distributed in Nonsan. The reaction solution was denatured at 94 ° C. for 5 minutes after adding extracted DNA to the manufacturer's premix kit, followed by 45 cycles of continuous reaction (modification: 30 seconds at 94 ° C., annealing: 30 seconds at 54 ° C., amplification: 40 seconds at 72 ° C.). 5 minutes at the last 72 ℃.

As a result, all of the PCV2 envelope protein gene 702 bp was synthesized in seven blood samples distributed in Nonsan (FIG. 2).

3. Purification of virus isolated from blood samples and analysis of envelope proteins

(1) Virus isolation and purification

Serum was separated by centrifugation of 40 ml of blood samples received at 14,000 rpm. To remove the host material, apply a thin layer of 30% celite545 (w / v) and 20% charcoal (w / v) to a Buchner funnel equipped with Whatman No.1, respectively, and apply serum on the surface. Filtered. PEG 8000 dissolved in phsophate-buffered saline (1x PBS; 137 mM NaCl, 2,7 mM KCl, 4,3 mM Na ₂ HPO ₄ , 1.4 mM KH ₂ PO ₄ , pH 7.2) for primary virus collection. Promega, USA) solution (40% PEG, 2.4% NaCl) was added to 4 times the virus filtrate and left for 1 hour at 4 ℃. After centrifugation at 10,000 rpm for 20 minutes, the precipitate is suspended with 1/10 times 1x PBS of the filtrate, and then 1/4 and 1/2 times the amount of chloroform or 7% n-butanol (v / v) of the precipitate suspension. Mixed. The mixed solution was centrifuged at low speed, and then the supernatant was taken and loaded on a 5-20% sugar solution (v / v) density gradient dissolved in 1x PBS. The tube was mounted on a SW41Ti rotor (Beckman Coulter Inc., CA, USA) and subjected to ultrafast centrifugation at 267,000 g for 18 hours to obtain a virus-containing fraction. To concentrate the virus, the fractions were diluted with 1x PBS (1: 1, v / v) and subjected to ultra-centrifugation using 10% sugar solution (v / v) as a cushion. The sugar solution density gradient centrifugation method was performed as above. A schematic diagram depicting the separated tablets is shown in FIG. 3.

(2) SDS-PAGE and Western blot analysis

In addition to the fractions obtained in the last step, a sample is obtained at each completion of the separation and purification process, and a modified sample (Laemmli, 1970, Nature 227: 680-685) is added, heated for 3-5 minutes, and then denatured. Was divided into two gels prepared and subjected to SDS-PAGE electrophoresis, respectively. After electrophoresis, one gel was stained with Coomassie blue solution (dissolved 0.05% Coommasie brilliant blue R-250 in a mixture of methanol, glacial acetic acid and water (5: 1: 5, v / v / v)). Proteins were analyzed and the gels were transferred to nitrocellulose membrane (NCM) by electrophoresis for immunological analysis without staining the other gels. NCM was immersed in a blocking solution of 5% fat-free milk powder in 1x PBS to suppress nonspecific reaction, treated for 1 hour on a stirrer and washed in 1x PBS buffer containing 0.03% Tween-20. The primary antibody was purchased from polyclonal antisera against PCV2 from the manufacturer (VMRD, Pullman, USA) and diluted with 1X PBS (1: 1,000, V / V). Goat anti-pig IgG alkaline phosphatase conjugate (Sigma Co., USA) was used after diluting (1: 30,000, v / v). Reactions with the primary and secondary antibodies were each treated for 1 hour and washed three times after each step. Finally, NCM was developed by treatment with a buffer solution containing nitroblue tetrazoilium (NBT) / 5-bromo-4-chloro-3-indolylphosphate (BCIP) (100 mM Tris, 100 mM NaCl, 5 mM MgCl ₂ , pH 9.5). I was.

In serum filtration ability, celite was more effective in removing hemoglobin and impurities such as hemachrome than charcoal. The optimal concentration of PEG, an aqueous precipitation aid, was 0.4% and 0.48% NaCl, respectively. In addition, in order to determine the efficacy of the organic solvent in removing host substances and impurities, the serum sample (PCU-1) was concentrated with PEG without filtering with celite, and the precipitate was dissolved in chloroform and n-butanol. After treatment, SDS-PAGE / Western blot was performed. Experimental results showed that both organic solvents were effective in purifying the host material and impurities. When the organic solvent was not treated, the envelope protein of PCV was observed on SDS-PAGE, but the band was not detected by Western blot analysis. This is believed to be because certain substances present in serum interfere with antigen-antibody specific responses. In addition, when chloroform was treated at 1: 1 ratio with the sample and 7% n-butanol was treated at 1: 1 ratio with the sample, the color reaction of Western blot due to the reaction between the PCV envelope protein and the antibody was observed. Was somewhat faint than that of the 1: 4 ratio with the sample. This is probably due to the damage to the surface of the viral envelope protein when treated with chloroform in a 1: 1 ratio with the sample and 7% n-butanol in a 1: 1 ratio with the sample (Fig. 4). Therefore, in order to verify the reproducibility of the experimental results, the experiments were performed on the five blood samples supplied from Nonsan (PCU-4, -5, -6, -7, -8) by the method described above. Envelope protein was clearly detected by the antibody (FIG. 5).

The blood sample (PCU-1) was filtered through celite, concentrated with PEG, and then treated with chloroform (Fig. 6). The host-derived protein was more effective than the PEG-treated and chloroform-treated samples without filtration (Fig. 4 and 5). Could be compared to eliminated. After performing the first sugar solution density gradient centrifugation method, the remaining cellular proteins were removed by the second sugar solution density gradient centrifugation method (FIG. 6).

The virus isolation and purification method devised from the above results can be applied to discriminate the infected virus from blood samples of humans or animals rather than time-consuming cell culture.

FIG. 1 is a photograph illustrating the DNA amplification of the envelope protein gene after PCR was performed at different concentrations of MgCl ₂ using PCV envelope protein-specific primers on blood samples collected from Okcheon. (Odd number: 1.5 mM, even number is 3.5 mM MgCl ₂ .M: 1Kb DNA ladder, PCU-3 (lane 1, 2), PCU-4 (lanes 3, 4), PCU-1 (lanes 5, 6) , PCU-2 (lanes 7, 8)

FIG. 2 is a photograph illustrating the DNA amplification of the envelope protein gene after PCR was performed at a concentration of MgCl ₂ 3.5 mM using the envelope protein-specific primer of PCV on blood samples collected from Nonsan. Positive control (lane 1), PCU-5, -6, -7, -8, -9, -10, -11 (lanes 2-8)

Figure 3 is a schematic diagram of a process for separating and purifying porcine circovirus from infectious blood according to the present invention.

FIG. 4 is a photograph of 12% SDS-PAGE (left) and Western blot (right) for the effect of organic solvent purification on blood samples (PCU-1). Virus suspension not purified with organic solvent (lane 1), chloroform treated with virus suspension 1: 1 (v / v) (lane 2), chloroform treated with virus suspension 1: 4 (v / v) (lane 3), 7% n-butanol treated with virus suspension 1: 1 (v / v) (lane 4)

FIG. 5 is PEG precipitated blood samples collected from Nonsan and the suspensions were purified by chloroform 1: 1 (v / v) treatment and analyzed by 12% SDS-PAGE (left) and Western blot (right). It is a photograph. Samples are PCU-4, -5, -6, -7, -8 (lanes 1-5)

Figure 6 is an analysis of 12% SDS-PAGE blood sample (PCU-1) was purified after celite filtration, PEG precipitation, chloroform purification sample (left, lane 1), primary sugar solution density gradient centrifugation Samples (left, lane 2), samples fractionated after the second sugar solution density gradient centrifugation (right, lanes 3-6), M: Page gel ladder (PAGE Ruler, Fermentas, USA)

Claims (3)

A process for purely separating and purifying infectious or unknown viruses from human or animal blood The method and apparatus for filtration using celite according to claim 1 According to claim 1, Purification method and apparatus using chloroform

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