No 2, a gene coding the same, and a method for producing the reverse transcriptase.

BACKGROUND A virus has a simple structure. That is, a virus has DNA or RNA in its center core'which is surrounded by a protein capsid. When a virus invades an animal or a plant, it uses an enzyme existed in the infected cell (host) to replicate DNA or RNA, to synthesize a protein and to proliferate.

Since a virus cannot proliferate by itself, it is, in fact, not an organism, but is often classified into a microorganism. A virus has two types; a DNA

virus and a RNA virus. A RNA virus has RNA as a genetic material in core. As a RNA virus infects a host, it produces DNA from RNA within a host and integrates the produced DNA to a chromosome of a host for replication. A RNA virus is called a 'retrovirus'because it has an enzyme inducing reverse transcription from RNA to DNA.

The replication of a retrovirus in a host cell is as follows; 1) RNA of the virus gets into a host cell, 2) RNA of the virus is used as a template for the synthesis of a corresponding DNA strand, and 3) the synthesized single DNA strand changes into double strand by an enzyme having an activity of synthesizing DNA. A retrovirus has a gene coding a reverse transcriptase, so that it induces reverse transcription from RNA to DNA by itself, making it possible to get into a chromosome of a host. Once a DNA is integrated into a chromosome of a host, it starts synthesizing a viral protein. With all the mentioned characteristics, a retrovirus can accept much more genetic materials than a DNA virus and can invade into a variety of living creatures. A retrovirus (such as HIV, HTLV-1 (human T-lymphocyte leukemia virus-1), etc.) has been known to cause immunodeficiency or tumors not only in animals but

also in human. Besides, a retrovirus used to be used as a vector carrying a gene into an animal or a plant cell.

A reverse transcriptase of a retrovirus synthesizes cDNA having corresponding base-sequences by using its RNA as a template, and is very important enzyme for analyzing RNA such as RT-PCR and DNA chip, etc. Thus, the enzyme has been widely used for gene cloning or diagnosing a disease by synthesizing cDNA by using RNA as a template (Mocharla et al., Gene, 1990, 99 : 271-275).

Therefore, the mass-production of the reverse transcriptase by inducing mass-expression in E. coli as a recombinant protein after cloning facilitates a gene cloning or diagnosis of a disease.

Plenty of vectors having a promoter, which is powerful in production of a recombinant protein in E. coli but is easily induced, have been developed for the production of a foreign protein. Though, as the recombinant protein was over expressed in E. coli, an insoluble consorte, known as an inclusion body, was often formed. The insoluble protein is easily separated from a water-soluble protein, and has an unnatural reduced form, resulting in a functional

inactivity. The inclusion body can be either beneficial or unprofitable depending on a biological process and the characteristics of a protein.

Precisely, as a recombinant protein induces an inclusion body in E. coli, early separation is easily acquired, making purification providing over 80% purity possible simply by centrifugation after cell crushing, and proteolysis caused by the attack of intracellular protease decreases. In the mean time, in order to change an inactive inclusion body into an active protein, denaturation & refolding processes are required, and further the purification process costs a lot owing to the low separation efficiency in the reformation of a structure. The formation of an inclusion body seems to be affected by a host-vector system, characteristics of a protein, culture and expression conditions, etc, but no general rules concerning the formation have been found yet.

If a recombinant protein is produced as a form of a water-soluble protein in cytoplasm, especially in case it is a protein that needs the formation of disulfide bond, the preparation of an expression system is very difficult and culture conditions are

troublesome, too. On the contrary, a recombinant protein is produced in the form of an inclusion body, the preparation of a vector and culture conditions are simple and easy, suggesting that HCDC (high cell density cultivation) can be used conveniently for mass-production.

When a target protein is produced in the form of an inclusion body, denaturation and refolding processes are necessary to change it into an active protein, during which purification efficiency might vary from the characteristics of a protein. Thus, it is very important to establish a purification procedure that can minimize the loss of a protein.

Reticuloendotheliosis, mostly developed in fowls, cocks turkeys and ducks, is caused by reticuloendotheliosis virus (REV) belonging to a retrovirus, and carries symptoms such as decrease of immunity, underdevelopment, acute and chronic tumors, etc. The reticuloendotheliosis has been reported in many countries including Australia, Japan, U. S. A. and England. In particular, an outbreak of the reticuloendotheliosis was once reported in Australia and Japan because of misuse of Marek's disease vaccine infected already with the

reticuloendotheliosis virus. The development of the reticuloendotheliosis was first reported in 1991 in Korea and the reticuloendotheliosis virus separated in Korea was confirmed to have a highly active pathogenicity causing serious decrease of immunity and gaining rate, and chronic tumors. Among the whole DNA base-sequences of pol gene in reticuloendotheliosis virus, just 3'-end and 5'-end regions have been disclosed so far.

Thus, the present inventors investigated the whole base sequence of pol gene in reticuloendotheliosis virus in order to provide a novel recombinant reverse transcriptase which is highly available in industries, by which a gene of the reverse transcriptase was cloned, resulting in the disclosure of the whole base sequence. And further, the present inventors completed the invention by developing a method to change an inactive transcriptase protein into an active form.

SUMMARY OF THE INVENTION It is an object of this invention to provide a reverse transcriptase of reticuloendotheliosis virus

and a gene coding the same.

It is also an object of this invention to provide a vector containing the gene above and a transformant including the same.

It is a further object of this invention to provide a method for the production of a reverse transcriptase of reticuloendotheliosis virus which becomes activated by a denaturation & refolding method changing an inactive recombinant reverse transcriptase of reticuloendotheliosis virus, which used to be generated in the form of insoluble protein as being mass-produced in a host cell, into an active protein.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS In order to achieve the above objects, the present invention provides a reverse transcriptase of reticuloendotheliosis virus and a gene coding the same.

The present invention also provides a vector containing the gene above and a transformant including the same.

The present invention further provides a method for the production of a reverse transcriptase of

reticuloendotheliosis virus which becomes activated by a denaturation & refolding method changing an inactive recombinant reverse transcriptase of reticuloendotheliosis virus, which used to be generated in the form of an insoluble protein as being mass-produced in a host cell, into an active protein.

Hereinafter, the present invention is described in detail.

The present invention provides a reverse transcriptase of reticuloendotheliosis virus represented by SEQ. ID. No 2 and variants thereof.

The present invention also provides a gene of the reverse transcriptase of reticuloendotheliosis virus represented by SEQ. ID. No 1, which encodes the reverse transcriptase of reticuloendotheliosis virus, homologs and variants of the same.

The"homologs (gene)"of the gene of the reverse transcriptase of reticuloendotheliosis virus means genes having a coding sequence of the reverse transcriptase of the virus represented by SEQ. ID.

No 2. Thus, all the genes that can produce the reverse transcriptase of reticuloendotheliosis virus represented by SEQ. ID. No 2 are included in the

category of the'homologs'.

The"variants (enzyme)"of the reverse transcriptase of reticuloendotheliosis virus means enzymes in which one or more amino acid residues of the reverse transcriptase of the virus represented by SEQ. ID. No 2 are deleted, added, inserted or replaced. The variants (gene) of the reverse transcriptase gene of the reticuloendotheliosis virus include all the genes that can encode the variants (enzyme) of the reverse transcriptase of the virus.

The variants (enzyme) above preferably have over 95% homology to the reverse transcriptase of reticuloendotheliosis virus represented by SEQ. ID.

No 2, and more preferably have over 98% homology.

The variants (gene) above preferably have over 95% homology to the reverse transcriptase of reticuloendotheliosis virus represented by SEQ. ID.

No 1, and more preferably have over 98% homology.

The primary structure of a retrovirus has LTR- gag-pol-env-LTR in that order, in which LTR (long terminal repeat) is a promoter, gag is coding a core protein, pol is coding a reverse transcriptase and env is coding a capsid protein. All the reverse

transcriptase of every retroviruses are synthesized by pol gene and have activities of synthesizing DNA directly from RNA, synthesizing DNA from DNA and decomposing RNA. Pol gene is transcribed as a part of a huge polyprotein gene composed of gag-pro-pol.

And transcriptions of gag and pol varies from a retrovirus, that is, a transcription is terminated by recognizing stop codon between gag and pol, a transcription starts again by frame shift or a transcription to a polyprotein is completed at one try by stop codon readthrough, followed by cutting by a protease of a virus (Monica et al., J. Biol.

Chem, 1985, 260 : 9326-9335; Luke et al., Biochemistry, 1990, 29 : 1764-1769; Le Grice et al., J.

Virolo., 1991, 65 : 7004-7007).

The present inventors had host cells infected with reticuloendotheliosis virus to proliferate the virus. Then, viral genome was extracted to investigate the base sequence of terminal parts of pol gene, known to code all the reverse transcriptase of each retrovirus. Based on that, common primers were prepared, by which the whole base sequence of the pol gene was analyzed. Then, a presumed reverse transcriptase of

reticuloendotheliosis virus was compared with those of other strains. Based on the information on the sequence of the pol gene obtained above, the reverse transcriptase of reticuloendotheliosis virus was presumed to be a monomer, and a primer of the reverse transcriptase was designed by the presumption. A reverse transcriptase having a sequence designed above was inserted into an expression vector of E. coli, leading to transformation of E. coli. Thus, a transformed E. coli strain was obtained by using a vector containing the above reverse transcriptase.

As a result, a novel gene of a reverse transcriptase of reticuloendotheliosis virus was confirmed to have a base sequence represented by SEQ.

ID. No 1, and the reverse transcriptase of reticuloendotheliosis virus transcribed by the above gene was confirmed to have an amino acid sequence represented by SEQ. ID. No 2. After comparing the base sequence of the above gene with other strains by BLAST SEARCH (National Center for Biotechnology Information; NCBI), reticuloendotheliosis virus was confirmed to be a retrovirus having 55% and 57% homology to MMLV (moloney murine leukemia virus) and FelV (feline leukemia virus), C-type retroviruses.

After comparing the amino acid sequence represented by SEQ. ID. No 2 obtained by sequencing analysis with other sequences possibly obtained from frame shift on pol gene, it was confirmed to be very similar to the amino acid sequences of MMLV reverse transcriptase produced by stop codon readthrough.

Therefore, it was also confirmed that the reverse transcriptase of reticuloendotheliosis virus was transcribed by stop codon readthrough, just like the reverse transcriptase of c-type virus MMLV (moloney murine leukemia virus).

By using SWISS-MODEL (protein modeling server made by Swiss Institute of Bioinformatics), the tertiary structure was estimated by sequences. The estimated structure was, then, compared with an already known tertiary structure of MMLV to judge the similarity. The location of cysteine residue is very important in the formation of the tertiary structure of a protein. From the comparison of sequences between a reverse transcriptase of MMLV and a reverse transcriptase of the reticuloendotheliosis virus of the present invention, it was confirmed that the locations of cysteine residues were corresponded. So, the tertiary

structure of the reverse transcriptase protein of the reticuloendotheliosis virus was confirmed to be very similar to that of MMLV.

The present invention also provides a vector containing a reverse transcriptase gene of reticuloendotheliosis virus and a transformant (KCTC 10511BP) transfected with the same.

For the construction of a vector of the present invention, conventional vectors for prokaryotes or eukaryotes could be randomly selected according to an expression target, and it is a common sense for the people in this field that choice is not limited to a specific vector. In addition, a size and a base sequence of a gene being inserted into a foreign gene insertion region of a vector can be varied with conventional techniques. A transformant containing the said vector can be prepared by inserting the said vector into a host cell, and the method for the insertion can also be one of conventional techniques including heat shock, electroporation, etc. Besides, a choice of a host cell for the preparation of a transformant is not limited to a specific cell, either, that is, both prokaryotes and eukaryotes are available. But, E.

coli is, in particular, preferable for a host cell herein.

In the preferred embodiment of the present invention, a recombinant vector was constructed by inserting a reverse transcriptase gene of reticuloendotheliosis virus represented by SEQ. ID.

No 1 into a vector for transformation, which was then, named pETREV-RT'.

The present inventors deposited an E. coli transformant, prepared by transfecting E. coli (BL21DE3) with a vector (pETRV-RT) containing a reverse transcriptase gene of reticuloendotheliosis virus of the present invention, at Korean Collection for Type Culture (KCTC) of Korea Institute of Bioscience and Biotechnology (KRIBB), on August 20, 2003 (Accession No: KCTC 10511BP).

The present invention further provides a method for the production of an active reverse transcriptase of reticuloendotheliosis virus, which is comprised of the following steps: (1) Separating an inclusion body including an expressed recombinant reverse transcriptase of reticuloendotheliosis virus; (2) Dissolving the separated inclusion body in

a denaturation buffer containing urea or guanidine chloride; and (3) Refolding the dissolved protein by reacting it in a renaturation buffer.

In this invention, an E. coli strain, transfected with a vector containing a reverse transcriptase of reticuloendotheliosis virus, was cultured, and the expressed reverse transcriptase protein was changed into a water-soluble active protein by the said method. Then, the activity of the enzyme was investigated and compared with conventional reverse transcriptases. A transformant producing a recombinant reverse transcriptase of reticuloendotheliosis virus was designed to produce the enzyme as a monomer. So, if the obtained reverse transcriptase was confirmed to have an activity, it will be determined to be a monomer.

As a result, an activity of a recombinant reverse transcriptase of reticuloendotheliosis virus, prepared by being through denaturation and refolding, was confirmed. While a reverse transcriptase protein of HIV or AMV is to be active in the form of a dimer (Eisenman et al., J. Virol, 1980, 36 : 62-78), the recombinant reverse transcriptase of

reticuloendotheliosis virus is to be active in the form of a monomer (see FIG. 3). Therefore, the method of the present invention facilitates the production of an active reverse transcriptase of reticuloendotheliosis virus since an inactive recombinant reverse transcriptase of reticuloendotheliosis virus can be easily changed into an active form of a water-soluble protein by the method.

BRIEF DESCRIPTION OF THE DRAWINGS The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein : FIG. 1 is a schematic diagram showing a vector pET21bREV-RT'used for the cloning of a reverse transcriptase of reticuloendotheliosis virus gene, FIG. 2 is an electrophoresis photograph showing the result of SDS-PAGE with a reverse transcriptase strain of reticuloendotheliosis virus each obtained from every stage from cell crushing to inclusion body separation,

Lane 1 : Protein size marker, Lane 2 : BL21DE3, Lane 3 : BL21DE3+pET21bREV-RT no induction, Lane 4 : BL21DE3+pET21bREV-RT induction, Lane 5 : BL21DE3+pET21bREV-RT induction/ water-soluble fraction, Lane 6 : BL21DE3+pET21bREV-RT induction/ insoluble pellet washed fraction, Lane 7 : BL21DE3+pET21bREV-RT induction/ insoluble inclusion body fraction, FIG. 3 is an electrophoresis photograph showing cDNA having an activity of a reverse transcriptase of reticuloendotheliosis virus by refolding conditions.

Lane 1 : DNA size marker, Lane 2 : 50 mM Tris + 1 mM GSSG + 10 mM GSH, Lane 3 : 50 mM Tris + 1 mM GSSG +10 mM GSH + Tween 20 0. 02%, Lane 4 : 50 mM Tris + 1 mM GSSG + 10 mM GSH + 5% sucrose, Lane 5 : 50 mM Tris + 1 mM GSSG +10 mM GSH +75 mM KC1, 3 mM MgCl2, Lane 6 : 50 mM Tris + 1 mM GSSG +10 mM GSH + PEG 8000 1 mg/L,

Lane 7 : 50 mM Tris + 1 mM GSSG +10 mM GSH + 25 mM CaCl2 + 25 mM MgCl2, Lane 8 : 50 mM Tris + 1 mM GSSG +10 mM GSH + 0. 1% BSA, Lane 9 : 50 mM Tris + 2 mM GSSG + 10 mM GSH, Lane 10 : 50 mM Tris + 2 mM GSSG +10 mM GSH + Tween 20 0. 02%, Lane 11 : 50 mM Tris + 2 mM GSSG + 10 mM GSH + 5% sucrose, Lane 12 : 50 mM Tris + 2 mM GSSG +10 mM GSH +75 mM KCl, 3 mM MgCl2, Lane 13 : 50 mM Tris + 2 mM GSSG +10 mM GSH + PEG 8000 1 mg/L, Lane 14 : 50 mM Tris + 2 mM GSSG +10 mM GSH + 25 mM CaCl2 + 25 mM MgCl2/ Lane 15 : 50 mM Tris + 2 mM GSSG +10 mM GSH + 0.1% BSA, Lane 16 : Negative control, Lane 17 : Positive control.

EXAMPLES Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Cloning of a reverse transcriptase gene of reticuloendotheliosis virus and analysis of the base-sequence of the same Chicken embryo filbrobalsts (CEFs) were infected with reticuloendotheliosis virus for the proliferation of the virus. Then, virus genome was extracted, from which a reverse transcriptase gene of reticuloendotheliosis virus was amplified by PCR using a primer synthesized based on pol gene whose sequences were analyzed earlier. As a result, a reverse transcriptase gene of reticuloendotheliosis virus was obtained. Particularly, a genome was extracted by following the instructions of manufacturer using G-DEX genomic extraction kit (iNtRON Co., Korea). 1 0 of the extracted genome was used as a template and 0. 5 M of 5 U/Taq polymerase (iNtRON co./Korea)/2 At of 10 X PCR buffer and 1 e of each primer (10 pmole),

represented by SEQ. ID. No 3 and No 4, were all mixed with 15. 5 of distilled water. The prepared reacting solution was denatured at 94 C for 5 minutes, followed by 30 cycles of denaturation at 94 for 30 seconds, annealing at 60 for 30 seconds, polymerization at 72C for 70 seconds, and final extension at 72C for 5 minutes. PCR product obtained above was electrophoresed on 1% agarose gel, followed by staining with EtBr (Ethidium Bromide) for observation.

The presumed size PCR product was observed after PCR using the primers above.

The reverse transcriptase gene obtained above was reacted with the vector pET21b (Novagen co., US), which were pre-treated with enzymes EcoRI and HindIII. After purification, they were linked by ligase. Particularly, 20 of PCR product was purified by using PCR quick-spin PCR product purification kit (iNtRON Co., Korea), to which pET21b vector and restriction enzymes EcoRI and HindIII were treated altogether. 10 of 3 M sodium acetate (pH 5.2) and 80 AQ of 95% ethanol were added to the enzyme treating PCR product and pET21b vector, respectively, which were then left at room

temperature for 5 minutes. Centrifugation was performed with 13, 000 rpm for 20 minutes, followed by washing with 70% ethanol. Precipitation was dried, then, dissolved again in 10 ß of triple- distilled water. 6 of the purified PCR product was mixed with 2 ß of the vector. 2 gt of 10X ligation buffer and 1 ß of ligase were added thereto, which was left at 16 C for a day. E. coli (BL21DE3) was transfected with the product by a general method such as calcium chloride method, etc. In the end, a transformant, in which the reverse transcriptase gene was inserted into the vector, was prepared.

The vector containing the reverse transcriptase gene above was investigated by enzyme cutting and sequence-analysis. Particularly, restriction enzymes EcoRI and HindIII were treated to the vector DNA obtained from the transformant, followed by electrophoresis. The expected size DNA fragment was observed. The vector, which was confirmed to include the reverse transcriptase, was named pET21bREV-RT' (FIG. 1). Base sequences of pET21bREV-RT vector were analyzed by using AB1377 DNA automatic sequencer and dye terminator kit (Perkin Elmer, Foster, CA), from which base sequences of the reverse transcriptase of

reticuloendotheliosis virus were determined. Amino acid sequences were also presumed therefrom.

As a result, the sequence was identified to be a base sequence represented by SEQ. ID. No 1 and the amino acid sequence translated by the above sequence was confirmed to be an amino acid sequence represented by SEQ. ID. No 2.

The present inventors deposited the E. coli transformant transfected with the vector containing the reverse transcriptase gene of reticuloendotheliosis virus of the invention at Korean Collection for Type Culture (KCTC) of Korea Institute of Bioscience and Biotechnology (KRIBB), on August 20, 2003 (Accession No: KCTC 10511BP).

Example 2: Expression of the reverse transcriptase of reticuloendotheliosis virus A transformant strain transfected with the vector containing the reverse transcriptase of reticuloendotheliosis virus obtained in the above Example 1 was cultured in the medium supplemented with the components shown in the below Table 1.

<Table 1> Composition of culture medium

Yeast Extract (Difco) 12.5 g NaCl 25 g Tryptone 25 g Ampicilin (50 mg/mQ in water) 250 ul Particularly, E. coli BL21DE3 strain having pET2lbREV-RT vector was cultured in the medium supplemented with the components shown in Table 1 for a day, then, 2 m of the culture solution was inoculated to express a reverse transcriptase of reticuloendotheliosis virus. During the cultivation, at the point of OD 0. 8, IPTG (Isopropyl ß-D- thiogalactopyranoside) was added to the medium at final concentration 1 Mm, followed by further culture for 3 hours. The IPTG treatment means the induction of a protein expression. Upon completing the culture, E. coli cells were centrifuged with 6,000 rpm at 4C for 10 minutes. 2 g of E. coli obtained above was dissolved in 5 m of cell lysis buffer A [50 mM Tris-HC1 (pH 8. 0), 1 mM EDTA (ethylendiamine tetraacetic acid), 25% sucrose, 2

mg/mQ lysozyme, 1 mM PMSF (phenylmethylsulfonyl <BR> fluoride) ], which was shook at 4°C for 30 minutes. 5 mg of cell lysis buffer B [50 mM Tris-HC1 (pH 8.0), 1% (v/v) Triton X-100,100 mM NaCl, 5 mM MgCl2] was added thereto, followed by another shaking at 4°C for 30 minutes. The cell lysate was centrifuged with 13,000 rpm for 10 minutes, resulting in the separation of supernatant including water-soluble proteins and cell inclusion body pellet including insoluble proteins. The obtained proteins were electrophoresed on SDS-polyacrylamide gel.

As a result, a reverse transcriptase of reticuloendotheliosis virus was detected in cell inclusion body pellet including insoluble proteins (FIG. 2). From the result, it was confirmed that a reverse transcriptase of reticuloendotheliosis virus was expressed in the form of an insoluble protein in the cell inclusion body, and was easily separated during the separation of the cell inclusion body.

Example 3 : Refolding of a reverse transcriptase of reticuloendotheliosis virus and confirmation of its activity The pellet containing a reverse transcriptase of reticuloendotheliosis virus of the Example 2 was dissolved in denaturation buffer (4 M GdnCl, 4 mM DTT, 50 mM Tris-HCl, and 1 mM EDTA (ethylenediaminetetraacetic acid), pH 8.0) by 20 mg/ m at 4 C for 1 hour. For the refolding, the solution was diluted in various renaturation buffers (20X volume), leading to a reaction at 4C for 24 hours. The compositions of the various renaturation buffers'used above were explained in Table 2.

<Table 2> Compositions of renaturation buffers 1 50 mM Tris + 1 mM GSSG + 10 mM GSH 2 50 mM Tris + 1 mM GSSG + 10 mM GSH + Tween20 0.02% 3 50 mM Tris + 1 mM GSSG + 10 mM GSH + 5% sucrose 4 50 mM Tris + 1 mM GSSG + 10 mM GSH + 75 mM KCl, 3 mM MgCl2 5 50 mM Tris + 1 mM GSSG + 10 mM GSH + PEG8000 1 mg/L 6 50 mM Tris + 1 mM GSSG + 10 mM GSH + 25 mM CaCl2 + 25 mM MgCl2 7 50 mM Tris + 1 mM GSSG + 10 mM GSH + 0.1% BSA 8 50 mM Tris + 2 mM GSSG + 10 mM GSH 9 50 mM Tris + 2 mM GSSG + 10 mM GSH + Tween20 0.02% 10 50 mM Tris + 2 mM GSSG + 10 mM GSH + 5% sucrose 11 50 mM Tris + 2 mM GSSG + 10 mM GSH + 75 mM KC1, 3 mM MgCl2 12 50 mM Tris + 2 mM GSSG + 10 mM GSH + PEG8000 1 mg/L 13 50 mM Tris + 2 mM GSSG + 10 mM GSH + 25 mM CaCl2 + 25 mM MgCl2 14 50 mM Tris + 2 mM GSSG +10 mM GSH + 0. 1% BSA

The activity of the refolded reverse transcriptase of reticuloendotheliosis virus was confirmed by RT-PCR with each reactant.

Particularly, cDNA was synthesized from each reticuloendotheliosis virus reactant by using a genome of ND virus (Newcastle Disease Virus), a kind of a RNA virus, as a template. The synthesized cDNA was amplified by PCR, followed by an electrophoresis (FIG. 3). As shown in FIG. 3,4 M guanidine chloride was used for the upper part of gel, and 6 M guanidine chloride was used for the lower part. An insoluble REV-RT was denatured and dissolved therein, and refolding was completed in the refolding buffer.

At last, the activity of the refolded reverse transcriptase of reticuloendotheliosis virus was

measured. By measuring the activity of a reverse transcriptase included in each reactant, the expected size DNA bands were confirmed.

As shown in FIG. 3, the expected size DNA bands were confirmed in many lanes on the agarose gel on which the PCR product was electrophoresed.

Therefore, the recombinant reverse transcriptase of reticuloendotheliosis virus, which was prepared by denaturation and refolding, was confirmed to have a normal enzyme activity. In particular, the darkest DNA band was seen in lane 4, suggesting that the recombinant reverse transcriptase of reticuloendotheliosis virus could have the most successful refolding process, that is, it could show the best activity when the composition of renaturation buffer No 4 in Table 2 was used.

INDUSTRIAL APPLICABILITY As explained hereinbefore, the reverse transcriptase of reticuloendotheliosis virus and the gene coding the same can be effectively used for the diagnosis of the infection with reticuloendotheliosis virus that decrease immunity and induces a tumor owing to its serious pathogenesis.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Manifestation on the Deposited Microorganism or Other Biological Materials A. The following manifestation is directed to the deposited microorganism or other biological materials described in the line 2 of page 10, the line 19 of page 10 and the line 15 of page 16 of the present application. B. Identification of the Microorganism An additional microorganism (s) is described in the next page (s) except the one Cl Depository Authority Korean Collection for Type Cultures (KCTC) Address (Zip code and Nationality) Korean Collection for Type Cultures (KCTC) Korea Research Institute of Bioscience and Biotechnology (KRIBB) #52, Oun-dong, Yusong-ku, Taejon 305 333, Republic of Korea Date Accession number August 20, 2003 KCTC 10511BP C. Additional Manifestation (filed as"blank"if there is no disclosure) To be continued to the additional next page Q D. Characteristic Items directed to the Manifestation E. Any Additional Manifestation (filed as"blank"if there is no disclosure) Any of the following manifestation will be filed to the International Bureau of the World Intellectual Property Organization later. (General Properties of the manifestation should be specifically described) Items for a Receiving Authority Items for the International Bureau D Accepted with the international D Accepted by the International application Bureau Authorized Official (s) Authorized Official (s)