KR20020020281A - Gene recombinant protein for diagnosis of tsutsugamushi disease - Google Patents

Abstract

PURPOSE: Provided is a genetic recombinant antigen protein for diagnosing tsutsugamushi disease, thereby increasing diagnostic sensitivity and reducing the cost of production. CONSTITUTION: The genetic recombinant antigen protein for diagnosing tsutsugamushi disease is characteristically a fusion protein of at least two kinds of proteins derived from epitope of Orientia tsutsugamushi. It is manufactured by culturing transformant strain transformed with a vector including a linker DNA linking genes of at least two kinds of protein derived from Orientia tsutsugamushi. It can be used for the diagnosing tsutsugamushi disease.

Description

Gene recombinant protein for diagnosing Tsutsugamu disease {GENE RECOMBINANT PROTEIN FOR DIAGNOSIS OF TSUTSUGAMUSHI DISEASE}

The present invention relates to a recombinant recombinant protein for diagnosing Tsutsugamus disease. Specifically, the present invention relates to a recombinant recombinant protein expressed in the form of a fusion protein by linking genes of the protein constituting the epitope of Orientia tsutsugamushi . It is related to the method of serological diagnosis of Tsutsugamushi disease, a species statutory infectious disease.

Tsutsugamus disease is caused by Orientia tsutsugamushi , a member of the Scrub typhus family of the genus Richecian , and the microorganisms present in the oral cavity are directly infected by humans when the larvae of the mite, which is a medium, inhale human tissue fluid Become onset. The symptoms are characterized by sudden fever after incubation, severe headache, papules, redness and bleeding.

Tsutsugamushi disease was reported for the first time in Korea by UN soldiers stationed in Korea (Munro-Faure, A, and Misseu MD: Scrub typhus in Korea.J. Of Roy Army Med. Corps., 97 : 227). (1954), Jackson et al. Collected hair mites from domestically captured rats and separated Orientia tsutsugamushi from 17% of Leptotrombidium pallidum , a kind of these mites. , EM, JX Danauskas, Smadel, JE et al .: Occurrence of Rickettsia tsutsugamushi in Korean rodents and chiggers.Amer . J. Hyg. 66 : 309-20, 1957). Since then, Twenty-seven patients with leuktospirosis and renal hemorrhagic fever among patients with febrile diseases in Jinhae Province have been diagnosed with Tsutsumashi disease (Lee Kang-soo, Jung-seop Kwon, Oh-hyun et al. 3). Rheumatic disease in Jinhae, Korea Journal of Microbiology, 21 : 113-120, 1986), and Lee Jung-sang also diagnosed Tsutsuma-shi disease in 9 patients with unknown causes (Lee Jung-sang, Ahn Kyu-ri, Kim Yoon-kwon and others: In Korea, a resident Korean) Rickettsia infection, including the first 9 confirmed cases of tsutsugamushi disease , Korean Medical Association, 29 : 430-438, 1986. In addition, in 1987, Jang and Kang reported the isolation of cells from Korean patients (Chang Woo-hyun and Kang Jae-seung: Isolation of Rickettsia tsutsugamushi from patients. Korean Medical Association, 30 : 999-1008, 1987).

Tsutsugamushi disease occurs worldwide, and especially there, including Malaysia, Thailand, the Philippines frequently in China, Japan and Australia (Traub, R. and CL Wisserman, Jr .: The ecology of chigger-borne rickettsiosis. J. Med Entem 11:.... ... 237, 1974; Rapmund, G., Upham, RW, Jr., Kundin, WD et al 2: Transovarial development of scrub typhus rickettsiae in colony of mites vector Trans Roy Soc Trop Med Hyg. 63 : 251, 1969; Shirai, A. and CL Wisserman, Jr .: Serological classification of scrub typhus isolates from Pakistan.Amer.J. Trop.Med . Hyg. 24 : 145, 1975; Shirai, A. Dohany. , E. and Gan, TC: Antigenic classification of Rickettsia tsutsugamushi isolated from small mammals traped in developing oil palm complex in peninsula Malaysia.Jap.J. Med.Sci.Biol . 33 : 231.1980; Zhang and Kang, 1987), domestically It is known that many patients with disease that have been called gangrene in rural areas are Tsutsugamus disease (Jang Woo-hyun, Ik-Sang Kim, Myung-Sik Choi and 18 others. Serological investigation of rash onset in Korea in 1987 and 1988. Korean Journal of Microbiology, 24 : 399-409, 1989). Tsutsugamushi disease mainly occurs in autumn, which is mainly related to the density of the chirping larvae, chigger, and from October to November when many larvae of Leptotrombidium pallidum appear in Korea. J. Korean Med. Sci., 31 : 601, 1988) There are many cases of Ttsugagamushi disease in Korea and the distribution of Rickettsia tsutsugamushi.

The hair mite that mediates Tsutsugamus disease is a type of arthropod belonging to the genus Leptotrombidium , and is free in nature. When the larvae of hair mites metamorphose, the tissue fluid of the vertebrate is needed and the parasitic parasites of the animal's body, the human is infected by the larvae of this time. Trombiculid mites collected in Korea are about 20 species (Traub, R., ML Morrow and LJ Lipovsky: New species of chiggers from Korea.Proc . Ent. Soc. Wash. 60 : 145-66, 1958), among which Orientia tsutsugamushi mediates Leptotrombidium pallidum and Leptotrombidium scutellaris .

Orientia tsutsugamushi is an absolute parasitic bacterium belonging to the genus Rickettsia. The genus Rickettsia is divided into typhus, spotted fever, and scrub typhus groups, including Rochalimea quintana and Q fever, which cause trench fever. Coxiella burnetti is added which causes fever. This classification is based on the host animal, the type of mediator that propagates the bacteria, the Weil-Felix test, and the response trends of the complement binding reactions, which have recently been identified by antigen analysis and molecular biology methods. It is becoming.

The bacteria belonging to the genus Rickettsia all cause disease in humans through insects such as ticks, teeth, fleas, etc. The symptoms and epidemiology of the disease are different in each group. The bacteria in the same group are serologically related, but there is no serological cross-reaction with the bacteria in the other groups. Orientia tsutsugamushi , a member of the scrub typhus group, is classified as a single species, but there are many serotypes with different antigen structures, and according to the differences of these antigens, Giliam, Kaf, Kato (Gilliam, Karp, Kato) et al. (Traub, R. and CL Wisserman, Jr., 1974). In addition to these three prototype strains, new serotypes such as TA678, TA686, TA716, TA763, and TH1817 were isolated in Asia (Shirai, A., Robinson, DM, Brown, GW et al .: Characterization of Rickettsia tsutsugamushi strains in two species of naturally infected laboratory-reared chiggers.Am. J. Trop.Med.Hyg. , 31 (2) : 395-402, 1982). Another Boryeong province was separated (Chang and Kang, 1987). According to this report, there are many differences in the serotypes of Orientia tsutsugamushi , which are separated from the suburbs of Gyeonggi-do and Chungcheong-do, which are located in the north of North Korea. Is to have.

Species-specific antigens and serotype-specific antigens are present in the cell membrane protein antigens of Orientia tsutsugamushi , which determines the variety of such serotypes. Orientia tsutsugamushi was purified and the antigens were analyzed by polyacrylamide gel electrophoresis and immunoblotting to reveal major antigens of 70, 60, 54-56, 46-47 kDa. (Takahashi, K., Urakami, H. and A. Tamura .: Purification of cell envelopes of Rickettsia tsutsugamushi. Microbiol. Immunol. 29 : 475, 1985), of which 46-47 kDa, 54-56 kDa and 70 kDa The proteins are antigenic, and the 70 kDa and 46-47 kDa proteins are strain specific antigens (Tamura, A., Urakami, H., and Tsurhara, T: Purification of Rickettsia tsutsugamushi by percol density gradient centrifugation.Microbiol . Immunol. 26 : 321, 1982).

Treatment of Orientia tsutsugamushi with trypsin loses its ability to penetrate mouse L cells, suggesting a close relationship between cell surface antigens and the pathogenicity of Orientia tsutsugamushi (Weiss, E .: The biology of Rickettsiae.Ann. Rev. Microbial. 36 : 345, 1982), the pathogenicity of Orientia tsutsugamushi , which differs between strains, differs in the structural differences of these type-specific proteins on the cell surface. (Tamura et al., 1982).

The pathogenicity of Orientia tsutsugamushi is due to systemic microvasculitis due to invasion of vascular endothelial cells. Orientation thiazol tsutsugamushi entered the blood (Orientia tsutsugamushi) is there is phagocytosis in the vascular cells by active phagocytosis, corrosion body (phagosome) is the multiplication by dichotomy the orientation thiazol tsutsugamushi (Orientia tsutsugamushi) glass into the cytoplasm from (Tamura et al., 1982) . Proliferated Orientia tsutsugamushi covers part of the host cell membrane and is released out of the cell, which later disappears (Tamura et al., 1982). Orientia tsutsugamushi ( orientia tsutsugamushi ) by the proliferation of vascular endothelial cells are destroyed or proliferation is triggered to cause micro clots and perivascular inflammation.

The mechanisms of defense against Orientia tsutsugamushi have not yet been fully understood, but the passive immunity of immune sera produced in other species of animals results in a protective effect in mice, and skin sensitization in humans or animals. It appears that both humoral and cellular immune mechanisms are involved. In nude mice infected with Orientia tsutsugamushi , antibodies were formed but did not have a protective effect against reinfection, and mice T lymphocytes immunized against reinfection of Orientia tsutsugamushi with different serotypes Along with the reports that adoptive delivery can provide passive defense (Shirai et al., 1982), the numerical association of T lymphocytes with the clinical picture of the patient is evidence that cellular immune mechanisms will work. On the other hand, reports that immune antibodies inhibited macrophage infections of Orientia tsutsugamushi by 50% suggest a related humoral immune response.

Immunity to Tsutsugamushi disease in humans is specific to one serotype. In other words, the protective immunity that develops after a disease lasts for years for the same serotype but only for months for other serotypes. For example, serologic studies have demonstrated that among patients observed for four months, they were initially infected with the Gilliam serotype and reinfected with the Karp serotype two months later.

The clinical symptoms of Tsutsuma's disease include sudden fever and headache, and there are generalized lymphadenopathy, crust formation and skin rash. In general, Tsutsugamus disease is recognized as a disease with mild clinical symptoms and easy clinical diagnosis. However, reports indicate that there are many cases of atypical clinical symptoms. Cases are difficult to differentiate from acute recessive diseases such as leptospirosis, typhoid fever and nephrotic hemorrhagic fever.

At present, the diagnosis of Tsutsugamushi disease is confirmed by isolation of Orientia tsutsugamushi from the patient's blood, the method of detecting antibodies against Orientia tsutsugamushi in the serum of the patient, and Polymerase Chain Reaction (PCR). There is a method of amplifying and testing DNA of Orientia tsutsugamushi in the blood of a patient using the method. Among them, the method of culturing the cells to diagnose the disease is not suitable for the purpose of diagnosis of the actual patient because the time required for the culture is more than 4 weeks, and the method using PCR is still in the development stage. Therefore, currently, the diagnosis of Tsutsugamus disease uses a serological method such as a Weil-Felix method, a complement binding reaction method, an indirect immunofluorescent antibody method, an immunoenzyme assay, and an immunoadhesive erythrocyte aggregation method.

However, among these serological methods, the Weil-Felix method is not only diagnosed with Tsutsugashi disease, but also with leptospirosis, urinary tract infections and other recessive diseases. It is not used. In addition, the complement binding reaction has the disadvantage that the test method is very technically demanding, and not only has low reproducibility, but also requires a purified Orientia tsutsugamushi antigen and a negative reaction when the antibody titer is low. Many are not widely used for the diagnosis of Tsutsuga-Mushi disease.

Therefore, indirect immunofluorescent antibody and immunoassay methods are the most widely used, and these two methods have high sensitivity and specificity, and can distinguish between IgG and IgM to distinguish early infection from reinfection. This is the preferred method for WHO. On the other hand, indirect immunofluorescent antibody test requires a facility for tissue culture or egg hatching in order to obtain the necessary antigen, and has the disadvantage that the test can only be performed in a laboratory equipped with a fluorescence microscope. Dilution as low as 1:10 or 1:20 has the disadvantage that a false positive reaction occurs. In addition, all of the methods used for such a serological diagnosis require a large amount of antigens, but there is a difficulty in that cell culture must be performed in order to culture bacteria and obtain antigens required for the test.

Accordingly, attempts have been made to isolate and purify genes of the Orientia tsutsugamushi main antigen in E. coli by genetic engineering and use them for diagnosis.

In the present invention, it is made in consideration of the above-mentioned difficulty in diagnosing the conventional Tsutsugamu disease, not only does not require the culture of the bacteria, but also increases the diagnostic sensitivity than using a single gene recombinant antigen, a single gene produced separately It is an object of the present invention to provide a recombinant fusion protein that can be used for diagnosing Tsutsugamushi disease that can reduce the production cost compared to using a recombinant protein and a method of producing the same.

FIG. 1 is a schematic diagram showing an example of serial binding of 56 kDa protein genes derived from three serotypes (Gilliam, Karp, Kato) of Orientia tsutsugamushi , where 1a represents vector pTGPT2 and 1b represents vector pETb7. ,

2 is a schematic diagram illustrating a process of constructing pTG3, pTGP1 and pTGPT2 vectors by sequentially introducing 56 kDa protein gene segments derived from three serotypes (Gilliam, Karp, Kato) of Orientia tsutsugamushi into pTYB12 vector,

FIG. 3 is a 56 kDa protein gene fragment derived from three serotypes (Gilliam, Karp, Kato) of Orientia tsutsugamushi , in which a pETb7 vector is produced by introducing a pET22b (+) vector from a pTGPT2 vector. Schematic diagram illustrating the process,

4 is an electrophoresis image of a vector of 56 kDa protein gene fragments derived from three serotypes (Gilliam, Karp, Kato) of Orientia tsutsugamushi in pTYB12 vector,

FIG. 5 is an electrophoresis image of a pETb7 vector cloned into a pET22b (+) vector of a 56 kDa protein gene fragment derived from three serotypes (Gilliam, Karp, Kato) of Orientia tsutsugamushi .

FIG. 6 shows electrophoresis showing the expression of 56 kDa protein genes derived from three serotypes (Gilliam, Karp, Kato) of Orientia tsutsugamushi cloned into pTYB12 vector,

FIG. 7 shows electrophoresis showing the expression of 56 kDa protein genes derived from three serotypes (Gilliam, Karp, Kato) of Orientia tsutsugamushi cloned into pET22b (+) vector,

FIG. 8 shows the isolation and purification of a fusion protein expressed in Escherichia coli transformed with a pTGPT2 vector in which a serial conjugate of 56 kDa protein genes derived from three serotypes (Gilliam, Karp, Kato) of Orientia tsutsugamushi was cloned. Showing the process,

9 is an enzyme immunoassay evaluating the reactivity of Tsutsugamushi disease patients with normal serum using a serial genetic recombinant antigen that is isolated and purified from a fusion protein produced by a serial conjugate of 56 kDa protein gene of Orientia tsutsugamushi . which,

Figure 10 shows the sensitivity and specificity of the enzyme immunoassay using a serial genetic recombinant antigen separated and purified fusion protein produced by the serial conjugate of the 56 kDa protein gene of Orientia tsutsugamushi ( Orientia tsutsugamushi ) that.

Tsutsugamus disease diagnostic protein of the present invention for achieving the above object is characterized by fusion of two or more proteins derived from the epitope of Orientia tsutsugamushi ( Orientia tsutsugamushi ).

Herein, the protein is preferably a fusion of a protein derived from 56 kDa protein of Orientia tsutsugamushi , and also, from a heterologous serotype strain of Orientia tsutsugamushi , in particular, Gilliam, It is preferred that the proteins derived from at least two species of Karp and Kato are fused.

In order to achieve the above another object, in the present invention, to produce a fusion protein for diagnosing Tsutsugamushi disease, comprising DNA connecting two or more genes derived from the epitope of Orientia tsutsugamushi . The present invention provides a method for producing a fusion protein for diagnosing Tsutsugamushi disease, comprising culturing a vector, and a transformant comprising the vector.

In the present invention, based on the known fact that 56 kDa protein of Orientia tsutsugamushi has antigenicity and is of diagnostic value, the standard serotypes of Giliam, Karp and Kato are 56 Genes encoding some fragments of the kDa protein were amplified by a polymerase chain reaction (PCR), serially linked to the amplified DNA fragments, cloned into a protein expression vector, expressed in E. coli and isolated. As a result, a fusion protein antigen that can be used for diagnosing Tsutsuga's disease can be produced and separated and purified simultaneously in a single process.

In other words, the gene of 56 kDa protein of the Orientia tsutsugamushi prototype strain showing strong reactivity with the serum of Tsutsugamushi disease patients was cloned to enable mass expression in Escherichia coli. This enables the development of passive hemagglutination reactions that can be easily used for the diagnosis of patients without the need for special testing instruments or equipment, and the use of methods such as ELISA, which is useful for the treatment of large-scale test materials such as epidemiological investigations.

Hereinafter, a process for preparing a fusion protein antigen for diagnosing Tsutsugamus disease of the present invention and using the same will be described in detail.

First, Gilliam, Karp, Kato and Boryong of Orientia tsutsugamushi are cultured in mouse L-929 cells, harvested and purified. After enzymatic digestion of the purified strain, DNA was purified by phenol extraction and ethanol precipitation.

Based on the nucleotide sequence of the Orientia tsutsugamushi 56 kDa protein gene, a protein site with an amino acid sequence of at least 30% homology between Boryungju and Giliam, Karp, and Kato serotypes By selecting, a pair of oligonucleotide primers are prepared in each of Gilliam, Karp, and Kato. With each of these primer pairs, a PCR reaction is carried out using Orientia tsutsugamushi DNA extracted above as a template to amplify the DNA fragments of Gillium, Kaf and Kato.

The PCR product is purified and cloned to link to the corresponding restriction enzyme cleavage site of the pTYB12 vector. First, a vector pTG3 is prepared by introducing a Gilt DNA fragment into a pTYB12 vector, a DNA fragment of a cap is introduced into pTG3 to prepare a vector pTGP1, and a vector pTGPT2 is prepared by introducing a Kato DNA fragment into pTGP1. In addition, a series linkage of DNA fragments of Gillium, Cap and Kato was cut from the vector pTGPT2 and introduced into the pET22b (+) vector to prepare a vector pETb7.

E. coli is transformed with the produced expression vector, and the transformed E. coli is cultured to induce fusion protein expression. After confirming by electrophoresis and Western blot, the expressed fusion protein antigen is isolated and purified and used for immunological quantitative and qualitative analysis.

Herein, immunological quantitative and qualitative analysis refers to qualitatively identifying antibodies against Orientia tsutsugamushi in human or animal serum to determine whether Orientia tsutsugamushi has a history of infection using isolated and purified proteins. And quantitative analysis techniques, and all methods capable of quantitative and qualitative analysis of antigen-antibody interactions developed so far, such as enzyme immunoassay, passive hemagglutination, and high density particle aggregation.

As described above, the enzyme immunoassay was used to evaluate the reactivity of Tsutsugamushi disease patients with normal serum using a serial gene recombinant antigen that was isolated and purified from a fusion protein produced by the serial conjugate of 56 kDa protein gene of Orientia tsutsugamushi . As a result, diagnostic sensitivity of 93.6% and diagnostic specificity of 94.2% were obtained.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only examples of the present invention, and the present invention is not limited thereto.

In addition, although these examples have described the fusion protein antigens produced by the serial conjugate of the 56 kDa protein gene derived from a specific strain of Orientia tsutsugamushi , the scope of the present invention is a medicine that exists worldwide. Fusion protein antigens produced by two or more series conjugates of protein genes constituting all protein epitopes, including 22, 47, 56, 58, 60, 110, 115, and 130 kDa proteins derived from more than 30 serotypes It will include up to. In addition, in the present embodiment, such a recombinant protein was produced by E. coli, but these proteins may be produced by cells such as yeast, other nucleated cells, animal or plant tissue cells, and recombinant mammals. It is included in the scope of the invention.

Example

One. Strains, Plasmids and Media

Strains from Orientia tsutsugamushi Boryeong, who are isolated from Tsutsugamu disease patients and stored in the Department of Microbiology, Seoul National University Medical School, and Giliam, Karp, and Kato (American Type) Culture Collection, Manassas, VA 20110-2209).

E. coli BL21 (DE3) and ER2566 were used for amplification and protein production of plasmids pCYB1, pCYB2, pCYB3, pCYB4, pTYB12 (NEB # 6902), pET (Novagen) and pBluescript (Stratagene), and plasmids.

E. coli transformed with the vector into which the gene fragments were introduced were cultured at 37 ° C. in LB (Luria-Bertani) liquid medium containing 100 μg / ml of ampicillin (Sigma A9518) or LB agar plate medium.

2. Orientia Tsutsugamu City Orientia tsutsugamushi Cultivation and Purification

Approximately 5 × 10 ⁷ mouse L-929 cells (American Type Culture Collection, Manassas, VA 20110-2209) contained 10% fetal calf serum (Gibco Lab. Grand island NY) in a 150 cm 3 cell culture vessel (Falcon 32025). Cultured using EMEM (Earles' Minimum Essential Media, Gibco 410-1500 EG, hereinafter abbreviated to EMEM-10) at 37 ℃ in the presence of 5% CO ₂ . When the cells proliferated and completely covered the basal surface of the culture vessel, all of the medium was removed leaving only 5 ml.

The suspensions of Giliam, Karp, Kato and Boryong of Orientia tsutsugamushi were placed in containers, respectively, and allowed to stand for 90 minutes in a carbon dioxide incubator at 37 ° C. EMEM (hereinafter abbreviated as EMEM-5D) containing 0.4 μg / ml of Daunomysin and 5% fetal calf serum was incubated at 34 ° C. in the presence of 5% CO ₂ . The cells were harvested when more than 80% of the cells were infected.

Orientia tsutsugamushi for attack for the defense of immunized mice was used without clarification and stored in liquefaction, and for stimulation of splenocytes and T-cells was isolated and purified by the following method: 20 Lycheechia-infected cells harvested from intestinal 150 cm 2 cell culture vessels were precipitated by centrifugation (Sorval) at 8,000 g for 15 minutes at 4 ° C., followed by 6.5 ml TS buffer (33 mM Tris-HCl pH 7.4, 0.25 M shoe Cross). Cells contained in the suspension were disrupted by a homogenizer (Potter-Elvehjem homogenizer) for 3 minutes and centrifuged at 400 g for 10 minutes to sink the cells. After adding percol to the supernatant to a final concentration of 40%, the lower layer of Orientia tsutsugamushi was harvested by isotropic centrifugation (SW41 Ti rotor, Beckman) at 25,000 g for 60 minutes, followed by phosphate buffer. Washed 6 times. After irradiating 600 krad with a radiation irradiator and stored at -70 ℃ was used as an antigen.

3. Orientia Tsutsugamu City Orientia tsutsugamushi Purification of DNA

Purified Orientia tsutsugamushi was buffered with 1% sodium dodecyl sulfate (SDS) and proteinase K (100 μg / ml) (10 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 150 mM NaCl) and dissolved at 50 ° C. for 2 hours. Add the same amount of phenol, mix slowly, and centrifuge at 12,000 g for 5 minutes to recover the supernatant (hereinafter abbreviated as phenol extraction), and in the same way phenol: chloroform: isoamyl alcohol (25: 24: 1 The protein was extracted and removed two more times using the mixed solution. 3 M sodium acetate (pH 5.4) was added so that the final concentration was 0.3 mM, and then mixed with twice the volume of 100% ethanol of the DNA solution and left at -20 ° C for 18 hours. DNA was precipitated by centrifugation at 12,000g for 15 minutes at 4 ° C. After discarding the supernatant, 70% ethanol was added to the same volume as 100% ethanol and washed by centrifugation for 15 minutes at 12,000g at 4 ℃. The supernatant was discarded and dried in a speed vacuum dryer (Savant SVC100-H) for 10 minutes, and then suspended in 30 μl of TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) and used. Abbreviated as ethanol precipitation).

4. Mouse Port Orientia Tsutsugamushi Orientia tsutsugamushi ) Preparation of Serum

The antiserum of C3H / HeDub mice against Orientia tsutsugamushi Boryeong for the cloning and Western blot analysis was prepared as follows: First, Orientia Tsutsugamushi cultured in mouse L-929 cells tsutsugamushi ) Boryeong strain 1000x MLD50 (measured in C3H / HeDub mice) was injected into the mice subcutaneously 0.2 ml each, and injected three times at two week intervals. Two weeks after the final immunization, the mice were sacrificed and the serum was harvested. The antibody titers of the Boryeong strains were measured by indirect immunofluorescence antibody, and the serum was collected at 1: 1280 or higher.

Antibodies against E. coli of the harvested serum were adsorbed and removed by the following method: E. coli transformed with the pTYB12 vector was incubated in 1 L of LB liquid medium containing 100 µg / ml of ampicillin for 18 hours, followed by tweening. Washed three times with tris-buffered saline (50 mM Tris-HCl, 300 mM NaCl, pH 7.4) containing 0.25% -20 and suspended in 20 ml TBST. After the bath was heated at 100 ° C. for 10 minutes, the cells were pulverized by treatment for 3 minutes at a maximum power in a sonicator (Quigley-Rochester Inc.). The mixture was mixed with the same volume of antiserum and adsorbed by shaking slowly at room temperature for 4 hours, followed by centrifugation at 2,000 g for 20 minutes to settle the cell debris and use the supernatant. Western blot analysis and immunological screening using this antiserum revealed a significant decrease in nonspecific reactivity.

5. Orientia Tsutsugamu City Orientia tsutsugamushi ) E. coli of 56 kDa protein

Expression and Separation Purification

(1) Amplification of 56 kDa Protein Gene Using Polymerase Chain Reaction

Based on Orientia tsutsugamushi 56 kDa protein gene Genbank accession number (L04956), Orientia tsutsugamushi Boryeong and Giliam, Karp, Kato By selecting sites showing high level similarity in all strains, a pair of oligonucleotide primers were prepared in each of Gilliam, Karp, and Kato. That is, in Gillium, to prepare a DNA fragment consisting of 127 to 1092 bases, the forward primer is 24 bases in the 127th to 3 'direction, and the reverse primer is in the 1092th to 5' direction. Each primer pair was prepared in a sequence complementary to 24 bases, and in the same manner, each primer pair for constructing a DNA fragment consisting of 118 to 1013 bases in a cap and a DNA fragment consisting of 121 to 957 bases in a Kato. (Oligo. Etc. Co. Wilsonville, OR.):

Gillium Potential Primer: 5 'GGA CA'T ATG ATT ACT GGT GCA GAA 3' (SEQ ID NO: 1)

Gillium Inversion Primer: 5 'ATC G'TC GAC ATT TAA AAG CCT AAC 3' (SEQ ID NO: 2)

CAP translocation primer: 5 'GTT G'TC GAC GGA ATG ATT ACT GGC 3' (SEQ ID NO: 3)

Cap inversion primer: 5 'GGC ATG ACA AAA TT G' AAT TC T ATC 3 '(SEQ ID NO: 4)

Kato potential primer: 5 'GTC GTT GGA G'AA TTC ATT ACT GGC 3' (SEQ ID NO: 5)

Kato inversion primer: 5 'TTG CTG CCC GGG' CCC CTG CTG 3 '(SEQ ID NO: 6)

The CA'TATG represents the restriction enzyme Nde I, the G'TCGAC is Sal I, the G'AATTC is EcoR I, and the GGG'CCC is Sma I.

Were also specific for the primer pair strain is verified by checking the orientation thiazol tsutsugamushi (Orientia tsutsugamushi) it is not responsive to extract DNA of uninfected L929 cells, DNA extracted from L929 cells orientation thiazol tsutsugamushi (Orientia tsutsugamushi) It carried out by the same method as DNA purification.

Using the Orientia tsutsugamushi DNA extracted above with each of these primer pairs as a template, PCR reactions were amplified according to the following method: orientia tsutsugamushi , namely Orientia tsutsugamushi. ) 100 ng of DNA was mixed in a PCR reaction mixture (400 nM each primer, 50 mM KCl, 10 mM Tris pH 9.0, 0.1% Triton X-100, 0.2 mM each of dNTP, 1 mM MgCl ₂ ) to the final 50 μl 2.5 U of Taq polymerase (Promega Cat # M1861, Madison, Wis.) Was added. PCR was carried out using a thermal cycler system (Thermal cycler system 9600, Perkin-Elmer Cetus, Norwalk, CT) once predenaturation for 3 minutes at 94 ℃, then 15 seconds at 94 ℃, 1 at 60 ℃ The minutes were repeated 35 times and the polymerization proceeded at 72 ° C. for 2 minutes after the last cycle.

Each amplified DNA was observed by UV irradiation after electrophoresis on an agarose gel containing ethidium bromide.

(2) Purification of PCR Products and Cloning into Expression Vectors

Each PCR product was isolated and purified according to instructions in the QIAEX gel extraction kit (Qiagen Inc., Catsworth CA.) and harvested with 20 μl of TE buffer. 400 ng of the purified PCR product was mixed in Klenow buffer (20 mM Tris-HCl, 100 mM MgCl ₂ , pH 8.0) and then Klenow fragment (Klenow fragment, Promega, M220, Madison, WI) 2.5 U Was added. After reacting at 37 ° C. for 3 minutes, 1/10 volume of dNTP mixture (0.125 mM each) was added, and the reaction was further performed at 37 ° C. for 1 hour. Phenol extraction and ethanol precipitation yielded 10 l of TE.

After the pTYB12 vector was isolated and purified, each PCR product obtained above was cloned into the restriction enzyme cleavage site. First, the pTYB12 vector and the Gillium PCR product were completely digested with restriction enzymes Nde I and Sac I, dephosphorylated with CIAP (Calf intestinal alkaline phosphatase), phenol extraction and ethanol precipitation, and harvested with 10 μl TE. 100 ng of the cleaved vector and 100 ng of the cleaved PCR product were mixed to 3.5 μl, treated at 45 ° C. for 15 minutes, and left on ice. To this, 0.5 μl of a 10-fold concentration of ligase buffer (250 mM Tris-HCl pH 7.8, 100 mM MgCl ₂ , 20 mM DTT, 4 mM ATP) and ligase 2 U were mixed and reacted at 4 ° C. for 18 hours. After gated, it was cloned into E. coli ER2566 and amplified. In this way, the introduction of the DNA fragment of Gillium into the pTYB12 vector was called pTG3.

In the same manner, the PCR products of pTG3 and CAP were digested with restriction enzymes Sal I and EcoR I, ligated and cloned into E. coli ER2566 for amplification. Thus, the introduction of the cap DNA fragment into pTG3 was called pTGP1.

Similarly, the PCR products of pTGP1 and Kato were digested with restriction enzymes EcoR I and Sma I, ligated and cloned into E. coli ER2566 for amplification. Thus, the introduction | transduction of the DNA fragment of Kato into pTGP1 was called pTGPT2.

In the pTGPT2 prepared as described above, the Gilt DNA fragment was 952 bps, the Capt DNA fragment was 873 bps, the Kato DNA fragment was 815 bps, and the total introduced DNA fragment was 2640 bps, about 97 kDa.

Next, the prepared pTGPT2 vector was digested with restriction enzymes Nde I and Sma I to obtain DNA fragments in which DNA fragments of Gillium, Cap and Kato were serially connected. The pET22b (+) vector was cleaved with Xho I, phenol extracted and ethanol precipitated, and then Klenau sections were added and dTTP and dCTP were added and reacted at 37 ° C. for 30 minutes. After the phenol extraction and ethanol precipitation treatment again, SI nuclease was added and reacted at 23 ° C. for 15 minutes, followed by phenol extraction and ethanol precipitation treatment. From this process, the Xho I cleaved part can be made into a smooth end. This was digested with Nde I, phenol extracted and ethanol precipitated, and then the vector was amplified by cloning into Escherichia coli ER2566 by ligation with chelium, cap and kato series linked DNA fragments. Thus, the introduction of DNA fragments in which the DNA fragments of Gillium, Kafe and Kato were serially introduced into pET22b (+) was called pETb7.

Positive clones were obtained by immunological search after transforming E. coli BL21 (DE3) with each prepared vector.

The vector pETb7 prepared by introducing a DNA fragment linked to a Gilt DNA fragment, a Capt DNA fragment and a Kato DNA fragment from the pTGPT2 vector to the pET22b (+) vector was transformed into Escherichia coli BL21 (DE3). Deposited as KCTC 18042P dated August 25, 2000.

FIG. 1 is a schematic diagram showing an example of serial binding of 56 kDa protein genes derived from three serotypes (Gilliam, Karp, Kato) of Orientia tsutsugamushi , where 1a represents vector pTGPT2 and 1b represents vector pETb7. .

FIG. 2 is a schematic diagram illustrating a process of constructing pTG3, pTGP1, and pTGPT2 vectors by sequentially introducing 56 kDa protein gene segments derived from three serotypes of Orientia tsutsugamushi (Gilliam, Karp, Kato) into pTYB12 vectors. to be.

FIG. 3 is a 56 kDa protein gene fragment derived from three serotypes (Gilliam, Karp, Kato) of Orientia tsutsugamushi , in which a pETb7 vector is produced by introducing a pET22b (+) vector from a pTGPT2 vector. A schematic diagram illustrating the process.

4 is an electrophoresis image of a vector of 56 kDa protein gene fragments derived from three serotypes (Gilliam, Karp, Kato) of Orientia tsutsugamushi , cloned into a pTYB12 vector, where M is the λHind III size label, V Is a pTYB12 vector, G is a pTG3 vector, P is a pTGP1 vector, and T is a pTGPT2 vector.

FIG. 5 is an electrophoretic image of a pETb7 vector obtained by cloning a 56 kDa protein gene fragment derived from three serotypes (Gilliam, Karp, Kato) of Orientia tsutsugamushi into a pET22b (+) vector, where M is λHind III Size markers, 1 represents the pET22b (+) vector (5493 bps) and 2 represents the pETb7 vector (8004 bps).

(3) Fabrication of competent cells.

Escherichia coli BL21 (DE3) shaken for 18 hours in LB medium was diluted 100-fold with LB medium and cultured until absorbance at 600 nm reached 0.3. In the case of the pTYB12 derivative vector and the pET7b vector, Escherichia coli ER2566, which was shaken for 18 hours in LB medium, was diluted 100-fold with LB medium and recultivated until absorbance at 600 nm reached 0.3. After standing in ice for 10 minutes, the supernatant was discarded by washing once with 0.1 M CaCl _{2 that} was stored in ice, and E. coli was suspended by adding 0.1 M CaCl ₂ to make 1/2 volume of the culture medium. After standing for 1 hour in ice and centrifuged at 4,000g for 20 minutes at 4 ℃, discarded the supernatant and suspended in 1/10 volume of 0.1 M CaCl ₂ of the culture medium was used for transformation.

(4) transformation.

1 μl of the expression vector prepared above and 100 μl of the competent cell were mixed and left to stand in ice for 1 hour, followed by thermal shock at 42 ° C. for 180 seconds. 500 μl of LB medium was added thereto, followed by incubation at 37 ° C. for 1 hour. After centrifugation at 8,000 g for 1 minute using a table-type microcentrifuge (MSE 41137.218) and discarding the supernatant, the cells were suspended in 200 μl LB, spread on LB agar plate medium, and incubated at 37 ° C.

(5) Immunological screening of transformed E. coli colonies.

The transformed Escherichia coli was spread and inoculated onto NC paper layered on LB agar medium and incubated at 37 ° C. for 18 hours. A colony-formed NC paper was placed on top of three stacks of 3 MM Whatman paper and a new NC paper was stacked on top of it. Three more 3 MM Whatman papers were overlaid and pressurized to produce duplicate NC papers. Each NC paper was placed on LB medium containing ampicillin (100 μg / ml) and incubated at 37 ° C. for 4 hours. Replicate NC papers were transferred onto LB agar plate medium containing 0.1 mM IPTG and ampicillin (100 μg / ml) and incubated for another 3 hours to induce protein expression.

After incubation, the NC paper was placed in a sealed glass bottle saturated with chloroform and exposed for 10 minutes. Lysozyme buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM MgCl ₂ , 3% bovine serum) Albumin (w / v), lysozyme 400 μg / ml, DNAase 1 U / ml) was dissolved by shaking slowly for 1 hour at room temperature. Gently wipe the NC paper surface and soak in fresh lysozyme buffer for 1 hour for the same method. After washing twice with TBST buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 10 minutes, the screening was performed with antiserum.

The NC paper was washed three times with TBST (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% Tween 20) and then reacted with 3% bovine serum albumin at room temperature for 30 minutes to block the margin area. Serum for Orientia tsutsugamushi Boryungju was diluted 1/40 with TBST, reacted with NC paper at room temperature for 30 minutes, and washed three times with TBST for 10 minutes. Secondary antibody (alkaline phosphatase conjugated anti-mouse IgG, Promega S3721) diluted 10,000-fold with TBST was reacted with NC paper for 30 minutes at room temperature and washed three times with TBST for 10 minutes each. Color substrate solution filtered with 3MM paper (100 mM Tris-HCl pH 9.5, 100 mM NaCl, 5 mM MgCl ₂ , nitroblue tetrazolium 0.3 mg / ml, 5-bromo-4-chloro-3) -Indolyl phosphate 0.15 mg / ml) was added to block the light and reacted for 15 minutes to obtain a positive clone.

(6) Polyacrylamide Gel Electrophoresis and Western Blot Analysis

Laemmli, 1970, modified the method as follows: 8% of acrylamide: bisacrylamide (30: 0.8) mixture in 0.37 M Tris-HCl (pH 8.8) solution containing 0.1% SDS was added. After the addition, remove air at a negative pressure, and add ammonium persulfate and N, N, N, N-tetramethylenediamine to 0.03% (w / v) and 0.066% (w / v), respectively, to cause a polymerization reaction. A resolving gel was made.

Acrylamide: bisacrylamide (30: 0.8) mixture was added to a 0.1% SDS and 0.125 M Tris-HCl (pH 6.8) solution to a final concentration of 5% to form a stacking gel, which was then separated. It was layered on a resolving gel.

The protein of E. coli to be electrophoresed was prepared by the following method: transformed E. coli was cultured for 18 hours, diluted 100-fold with medium, and absorbed at 0.1. When the absorbance reached 0.5, 0.1 mM IPTG was added and further cultured for 3 hours. After centrifugation at 4,000 g for 20 min at 4 ° C, the supernatant was discarded and washed once with 50 mM Tris-HCl (pH 7.4). Discard the supernatant and float the precipitated E. coli in sample buffer (2% SDS, 5% mercaptoethanol, 20% glycerol, 0.001% bromophenol blue (w / v), 0.1 M Tris-HCl pH 6.8) The bath was warmed for 5 minutes at. After centrifugation at 10,000 g for 10 minutes at room temperature, the supernatant was recovered and electrophoresed at 10 μl per well.

After the electrophoresis gel was stained or immunoblotting to confirm the protein. For dyeing, the gel was immersed in a dye solution (water: methanol: acetic acid (5: 5: 2), 0.1% (w / v) Coumasie Blue R250) for 1 hour, and then decolorized (25 % Methanol, 10% acetic acid) for 2 hours.

For immunoblotting, the gel and NC paper were overlaid and transferred at 90 V for 1 hour. Transcribed NC paper detected the protein by immunological search method.

FIG. 6 shows electrophoresis showing the expression of 56 kDa protein genes derived from three serotypes (Gilliam, Karp, Kato) of Orientia tsutsugamushi cloned into pTYB12 vector. Herein, V is a Gilliam 56 kDa protein fragment expressing E. coli ER2566 transformed with a pTYB12 vector, G is a pTGB vector, and P is a Gilliam 56 kDa protein expressing E. coli ER2566 transformed with a pTGP1 vector. Fusion protein of fragment and Karp 56 kDa protein fragment, T is a Giliam 56 kDa protein fragment, Karp 56 kDa protein fragment and Kato 56 expressed by E. coli ER2566 transformed with pTGPT2 vector The fusion protein of the kDa protein fragment is shown.

FIG. 7 shows electrophoresis showing the expression of 56 kDa protein genes derived from three serotypes (Gilliam, Karp, Kato) of Orientia tsutsugamushi cloned into pET22b (+) vector. Here, 1 is a fusion protein of Gilliam 56 kDa protein fragment, Karp 56 kDa protein fragment and Kato 56 kDa protein fragment expressing E. coli ER2566 transformed with pTGPT2 vector, and 2 is pET22b ( +) For E. coli ER2566 transformed with a vector, and 3 is a Giliam 56 kDa protein fragment, a Karp 56 kDa protein fragment and a Kato 56 kDa expressed by E. coli ER2566 transformed with a pETb7 vector. The fusion protein of the protein fragment is shown.

(7) Isolation and Purification of Recombinant Antigens

E. coli transformed with pTGPT2 was incubated at 4-37 ° C. for 18-72 hours and then diluted 100-fold with medium. When the absorbance at 600 nm reached 0.5, 0.1 mM IPTG was added and shaken for another 3 hours. Incubated. After centrifugation at 4 ° C. at 4,000 g for 15 minutes, with lysis buffer (50 mM Tris-HCl, 30 mM NaCl, 0.2% Tween 20, 10 mM-mercaptoethanol, 10 mM EDTA, 10 mM EGTA pH 7.0) Wealthy Lysozyme (1 mg / ml) was mixed and allowed to stand for 30 minutes in ice to melt E. coli. The supernatant was recovered by grinding for 1 minute at 50% of the maximum power with an ultrasonic mill and then centrifuging at 9,000g for 30 minutes at 4 ° C. In the same way, the protein was eluted three more times from the precipitate and the supernatant was mixed and used for separation. After adding the same amount of column buffer containing 0.25% Tween 20, NaCl concentration and pH were adjusted to 0.5 M and 7.0, respectively, and used for separation purification.

2 g of affinity chromatography resin (chitin, etc.) were mixed in 60 ml of column buffer (50 mM Tris-HCl, 0.5 M NaCl, 10 mM mercaptoethanol, 1 mM EDTA, 1 mM sodium azide) at room temperature Rehydration for minutes. The 2.5 × 50 cm column was filled with rehydrated resin and washed with three times the volume of column buffer. The E. coli lysate was passed through at a rate of 1 ml / min, followed by two volumes of washing with column buffer containing Tween 20 (0.25%) and two more volumes with column buffer alone.

Protein was eluted while measuring the absorbance of the eluate at 280 nm, and fractions were collected by 2 ml. Ultraviolet absorbance measurement (protein concentration (mL / mL) = 1.55 × E280-0.77 × E260), which collects 10 fractions with the highest absorbance and measures the absorbance at 280 nm and 260 nm, respectively, Protein was quantified.

8 is a process for separating and purifying a fusion protein from an E. coli transformed with a pTGPT2 vector in which a series conjugate of Orientia tsutsugamushi Gilliam, Karp, and Kato 56 kDa protein genes was cloned. It is shown. Here, the numbers indicate the sequence of fractions, and each fraction eluted was immunoenzyme stained with mouse antiserum after dot-blotting.

Ⓑ In the case of Escherichia coli transformed with pETb7, recombinant proteins were isolated and purified by culturing as follows: E. coli ER2566 transformed with pETb7 was cultured in LB medium containing ampicillin, and then cultured by diluting 100-fold, 600 Incubation was made until the absorbance at nm became 0.6, and IPTG was added so that the final concentration was 0.3 mM, followed by further incubation at 20 ° C for 24 hours. The cultured cells were centrifuged at 5,000 g for 30 minutes, then the supernatant was discarded and the precipitate was resuspended in binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). After sonication (sonification) the protein was extracted and centrifuged for 30 minutes at 12,000g was used for supernatant purification.

The amount of resin (His-resin) required for separation and purification was about 100 ml to 2.5 ml, and an appropriate amount of resin was placed in a column and allowed to equilibrate. The sterilized distilled water was flowed at least 3 times of the resin volume, and then the charge buffer (30 mM NiSO ₄ ) was flowed at least 5 times of the resin volume to allow the resin to charge. The equilibrium was then reached with a sufficient amount of binding buffer. The above E. coli suspension was added thereto and flowed over 10 times the binding buffer of the resin volume to remove nonspecific binding sites. The wash buffer (60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) of about 6 times the volume of the resin was then flowed to remove the remaining nonspecific binding sites. After almost flushing the wash buffer, elution buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) was added and the fractions collected. The eluted resin can be reused after flowing the strip buffer (100 mM EDTA, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) at least three times the resin volume to remove the charge by Ni.

6. Enzyme immunoassay

The recombinant fusion protein antigen obtained above was diluted to 5 nM in 0.05 M carbonate buffer (pH 9.6), and then 200 μl of the polyvinyl chloride plate (TiterTek, flat bottom, softplate) for EIA was injected 4 The reaction was carried out at 18 ° C. for 18 hours. Tween 20 was washed three times with 0.05% phosphate buffer (hereinafter referred to as PBST) and blocked with a 5% skim milk for 1 hour at room temperature.

The serum was diluted 1: 100, reacted for 30 minutes at room temperature, and washed six times with PBST. Secondary antibodies (horseradish peroxidase-covalently bound anti-mouse IgG, horseradish peroxidase-covalently bound anti-mouse IgM: Jackson 109-035-003) were diluted 10,000-fold with PBST at room temperature. After reacting for 30 minutes, the mixture was washed 6 times with PBST. Color development was performed with a chromogenic substrate solution (citric acid phosphate buffer, 0.4 μl / ml H ₂ O ₂ , 0.4 mg / ml o-phenylenediamine) for 15 minutes, and the color reaction was stopped with 2M H ₂ SO ₄ , followed by ELISA (enzyme-linked). Absorbance was measured at a wavelength of 492 nm with an immunosorbent assay reader (Dynatech MR 700).

9 is an enzyme immunoassay evaluating the reactivity of Tsutsugamushi disease patients with normal serum using a serial genetic recombinant antigen that is isolated and purified from a fusion protein produced by a serial conjugate of 56 kDa protein gene of Orientia tsutsugamushi . It is. The patient was diagnosed by an indirect immunofluorescence antibody method, in which the horizontal axis represents the antibody titer of serum measured by the indirect immunofluorescence antibody method, and the vertical axis represents the absorbance of the enzyme immunoassay.

Figure 10 shows the sensitivity and specificity of the enzyme immunoassay using a serial genetic recombinant antigen separated and purified fusion protein produced by the serial conjugate of the 56 kDa protein gene of Orientia tsutsugamushi ( Orientia tsutsugamushi ) It can be seen that a diagnostic sensitivity of 93.6% and a diagnostic specificity of 94.2% can be obtained.

As described above, the method for diagnosing Tsutsugamushi disease using the recombinant protein antigen according to the present invention corresponds to an indirect immunofluorescence antibody method in which an Orientia tsutsugamushi pathogen is cultured and used as a diagnostic antigen. Has diagnostic sensitivity and specificity. Therefore, according to the present invention, not only does not require pathogen culture, which is the limit of the conventional formulation of Tsutsugamus disease, but also increases the diagnostic sensitivity compared with the case of using a single recombinant protein, and a single gene produced separately. It is possible to economically obtain a good diagnostic formulation that can reduce the production cost compared to using a recombinant protein mixture.

Claims (13)

A protein for diagnosing Tsutsugamushi disease, characterized by fusion of two or more proteins derived from the epitope of Orientia tsutsugamushi . The protein of claim 1 wherein the protein is derived from a 56 kDa protein of Orientia tsutsugamushi . The protein according to claim 1 or 2, wherein the protein derived from a heterologous serotype strain of Orientia tsutsugamushi is fused. 4. The protein of claim 3, wherein the protein is derived from at least two species of Orientia tsutsugamushi from Gilliam, Karp, and Kato. A vector for producing a fusion protein for diagnosing Tsutsugamushi disease, comprising DNA linking two or more genes derived from an epitope of Orientia tsutsugamushi . 6. The vector according to claim 5, comprising DNA linking genes of proteins derived from 56 kDa proteins of Orientia tsutsugamushi . 7. The vector according to claim 5 or 6, comprising DNA linking genes of proteins derived from heterologous serotype strains of Orientia tsutsugamushi . 8. The vector according to claim 7, wherein the vector comprises DNA linking genes of proteins derived from at least two species of Orientia tsutsugamushi of Giliam, Karp, and Kato. . The vector pTGP1 of claim 8. The vector pTGPT2 of claim 8. The vector pETb7 (KCTC 18042P) according to claim 8. A method for producing a fusion protein for diagnosing Tsutsugamus disease, comprising culturing the transformant comprising the vector of claim 5. 13. The method of claim 12, wherein the transformant is at least one of E. coli, yeast, nucleated cells, animal tissue cells and plant tissue cells.