JP3831035B2 - Hepatitis C virus sensitive recombinant hepatocytes and use thereof - Google Patents

Description

【００３０】
実施例５：ＨＣＶ感染実験（その２）
ＮｍｙｃＮＳ５Ｂ−３の組換え肝細胞を用いてＨＣＶの感染実験を行った。感染材料を組換え細胞に添加した後、３７℃ＣＯ₂インキュベーター内で４時間吸着させた以外は、全て実施例４と同様の方法で行った。５６日間の観察経過を表２に示す。●がＨＣＶ陽性を、○がＨＣＶ陰性を表す。その結果、５６日間にわたりＨＣＶが検出された。ＮＳ５Ｂ遺伝子を導入していないＨｅｐＧ２細胞では、２８日間の観察期間にはＨＣＶは検出されなかった。
【００３１】
【表２】

【００３２】
実施例６：Ｉｎ ｓｉｔｕ ハイブリダイゼーション
Ｉｎ ｓｉｔｕハイブリダイゼーションによる細胞内ＨＣＶ ＲＮＡの検出を行った。
１０％ＦＢＳを含むウイリアムＥ培地に懸濁した感染細胞を、４ウェルＬａｂＴｅｋ Ｃｈａｍｂｅｒ（ヌンク社）の各ウェルに５ｘ１０⁴個播種し、３７℃で一夜吸着させた。翌日、パラホルムアルデヒドで細胞を固定した後、０．２μｇ／ｍｌのｐｒｏｔｅｉｎａｓｅ Ｋを５分間作用させた。この試料にジゴキシゲニン標識ＲＮＡプローブを加えて、５５℃で一夜ハイブリダイゼーションを行った。ＨＣＶゲノムを検出するためのプローブは以下のようにして調製した。まず、ＨＣＶの翻訳開始コドンである３４２番目の塩基より７００番目の塩基までの３５９塩基をＰＣＲで増幅した。その際、下流側のプライマーにはＴ７ ＲＮＡポリメラーゼの認識配列を付加しておいた。このようにして増幅された配列表の配列番号：８に記載のＨＣＶＤＮＡをＲＮＡプローブ合成の鋳型として用いた。ＲＮＡプローブの合成は、Ｔ７ ＲＮＡポリメラーゼ（ベーリンガー社）及び試薬を用いて、添付のプロトコールに従い、ジゴキシゲニン標識法で行った。
【００３３】
ハイブリダイゼーション後、同じく５５℃で５ｘＳＳＣ（２０ｘＳＳＣを４倍希釈したもの。２０ｘＳＳＣは１７５.３ｇのＮａＣｌと、８８.２ｇのクエン酸ナトリウムをＨ₂Ｏで１リットルとしてｐＨ７.０に調製したもの）液で約１分、更に２ｘＳＳＣで２０分、引き続き０.２ｘＳＳＣで２０分を２回の洗浄操作を行った。洗浄操作終了後、アルカリフォスファターゼ標識抗ジゴキシゲニン抗体（ベーリンガー社）を室温で３０分反応させた。次に基質溶液のＢＣＩＰ３３μｌおよびＮＢＴ４４μｌを加えて、室温にて６時間あるいは一夜反応させた。反応後、アセトカルミン液で細胞を染色して脱水した後、封入して光学顕微鏡で観察した。
【００３４】
実施例７：蛍光抗体法
１０％ＦＢＳを含むウイリアムＥ培地に懸濁した感染細胞を、４ウェルのＬａｂ Ｔｅｋ Ｃｈａｍｂｅｒ（ヌンク社）の各ウェルに５ｘ１０⁴個播種し、３７℃で一夜吸着させた。翌日８０％の冷エタノールで細胞を固定した後、常法に従い蛍光抗体反応を実施した。一次抗体にはＨＣＶゲノムのコア蛋白を大腸菌で発現させて精製した抗原を、マウスに免疫して調製したモノクローナル抗体を用いた。このモノクローナル抗体を１０μｇ/ｍｌの濃度で一次抗体反応を行った。二次抗体にはＦＩＴＣ標識抗マウスＩｇＧ（カッペル社）を用いた。
【００３５】
実施例８：アンチセンスオリゴヌクレオチドによるＨＣＶの複製阻害
１０％ＦＢＳを含むウイルアムＥ培地に懸濁したＮｍｙｃＮＳ５Ｂ-３細胞を２４ウェルプレートにウェル当り５ｘ１０⁴播種し、３７℃で培養した。培養液の容量は１ｍｌとした。２日後、細胞をＰＢＳで洗浄し、２％のＦＢＳを含むウイリアムＥ培地に置換した。次に、配列表の配列番号１９に記載の、ホスホロチオエート結合を骨格に持ち、ＨＣＶの塩基配列に対し、相補であるオリゴヌクレオチド（グライナージャパン社）および、同じ配列を持ち、３’にコレステロールを付加したオリゴヌクレオチドを、終濃度が前者の場合１０μＭ、後者の場合１μＭとなるように添加して１日培養した。陰性対象としてＨＣＶとは無関係の配列を有し、その他の構成要素は同じであるオリゴヌクレオチドを用いて、同様に処理した。翌日細胞をＰＢＳで２回洗浄し、ＨＣＶを感染させた。ＨＣＶの感染は感染材料５２６９および７８７８の各血漿を当量混合して使用し、３７℃で２時間吸着させた点以外は実施例４に記載の方法で行った。ウイルス吸着後、細胞をＰＢＳで５回洗浄し１０％のＦＢＳを含むウイリアムＥ培地を１ｍｌ添加して培養を開始した。感染材料接種３日後に感染細胞をオリゴヌクレオチドで再度処理した。すなわち、感染細胞をＰＢＳで１回洗浄後、２％のＦＢＳを含むウイリアムＥ培地１ｍｌに置換し、これに上述した濃度となるようにオリゴヌクレオチドをそれぞれ添加した。翌日終濃度が１０％となるように８０μｌのＦＢＳを加えて、培養を継続した。更に４日後に同じヌクレオチド処理操作を繰り返した。この３度目のオリゴヌクレオチド処理から４日後に培養上清および細胞を回収し、実施例４に記載の方法でＨＣＶＲＮＡの検出を行った。
【００３６】
その結果を図８に示す。図の番号１、２は正常ヒト血清の結果を示す。３〜６はオリゴ処理したＨＣＶ感染ＮｍｙｃＮＳ５Ｂ-３細胞の結果を示す。３は配列番号：１９のオリゴヌクレオチドによる処理、４は陰性対象のオリゴヌクレオチドによる処理、５は３’端にコレステロールを付加した配列番号：１９のオリゴヌクレオチドによる処理、６は３’端にコレステロールを付加した陰性対象のオリゴヌクレオチドによる処理をそれぞれ示す。７はオリゴヌクレオチド未処理のＨＣＶ感染ＮｍｙｃＮＳ５Ｂ-３細胞を示す。８はオリゴ未処理のＨＣＶ感染ＨｅｐＧ２細胞を示す。９はＨＣＶ非感染のＮｍｙｃＮＳ５Ｂ-３細胞を示す。１０はＨＣＶ陽性血漿の結果を示す。配列番号：１９のオリゴヌクレオチド処理を行った場合にＨＣＶの複製阻害効果が見られた。
【００３７】
【配列表】
配列番号：１
配列の長さ：３０
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列

【００３８】
配列番号：２
配列の長さ：３３
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列

【００３９】
配列番号：３
配列の長さ：４２
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列

【００４０】
配列番号：４
配列の長さ：４２
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列

【００４１】
配列番号：５
配列の長さ：３６６
配列の型：核酸
鎖の数：２本鎖
トポロジー：直鎖状
配列の種類：ｃＤＮＡ ｔｏ ｇｅｎｏｍｉｃ ＲＮＡ
他の情報：ＨＣＶゲノム１ｂ型の塩基配列の９０４１〜９４０６番目に相当する。
配列

【００４２】
配列番号：６
配列の長さ：２０
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列

【００４３】
配列番号：７
配列の長さ：２０
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列

【００４４】
配列番号：８
配列の長さ：２０
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列

【００４５】
配列番号：９
配列の長さ：２０
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列

【００４６】
配列番号：１０
配列の長さ：２１
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列

【００４７】
配列番号：１１
配列の長さ：２１
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列

【００４８】
配列番号：１２
配列の長さ：２１
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列

【００４９】
配列番号：１３
配列の長さ：２１
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列

【００５０】
配列番号：１４
配列の長さ：２０
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列

【００５１】
配列番号：１５
配列の長さ：２０
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列

【００５２】
配列番号：１６
配列の長さ：２０
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列

【００５３】
配列番号：１７
配列の長さ：２０
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列

【００５４】
配列番号：１８
配列の長さ：３５９
配列の型：核酸
鎖の数：２本鎖
トポロジー：直鎖状
配列の種類：ｃＤＮＡ ｔｏ ｇｅｎｏｍｉｃ ＲＮＡ
他の情報：ＨＣＶゲノム１ｂ型の塩基配列の３４２〜７００番目に相当する。
配列

【００５５】
配列番号：１９
配列の長さ：２０
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
他の情報：ＨＣＶゲノム１ｂ型の塩基配列の３４０〜３５９番目に相当し、これに相補的な配列で、ホスホロチオエート結合を骨格に持つ。
配列

【図面の簡単な説明】
【図１】ＨＣＶゲノムの遺伝子構造を示す図である。
【図２】発現ベクターｐＲｃ／ＣＭＶにＨＣＶの非構造タンパク質をコードする遺伝子領域を含むＤＮＡ断片（６．５ＨＣＶ）を挿入した発現プラスミドｐＲｃＨＣＮＳ６．５の構築の概略図を示す。
【図３】発現ベクターｐＧＳＴ−Ｅ／ＣＡＧにｍｙｃタグを挿入した発現プラスミドＮ−１２の構築の概略図を示す。ｍｙｃの１０アミノ酸を含む１２アミノ酸をコードするオリゴヌクレオチドと、これの相補鎖を合成し、アニールさせた後、発現ベクターのプロモーターの下流に挿入した。
【図４】ＨＣＶのＲＮＡポリメラーゼをコードする遺伝子を含むＤＮＡ断片（１．７７ＮＳ５Ｂ）を作製するための概略図を示す。
【図５】図３のＮ−１２のｍｙｃタグの下流に図４の１．７７ＮＳ５Ｂを挿入した発現プラスミドｐＧＣＮｍｙｃＮＳ５Ｂの構築の概略図を示す。
【図６】ｐＲｃＨＣＮＳ６.５を導入した肝細胞ＨｅｐＧ２Ｄｅｃ４、及びｐＧＣＮｍｙｃＮＳ５Ｂを導入したＮｍｙｃＮＳ５Ｂ−３をＳＤＳ−ＰＡＧＥにかけ、ウエスタンブロット法でＨＣＶ遺伝子に由来するタンパク質の発現を同定した結果を示す模式図である。
【図７】ＲＴ−ＰＣＲに用いたプライマーのＨＣＶゲノム上の位置を示す図である。Ａ領域、Ｂ領域あるいはＣ領域を用いて、ＲＴ−ＰＣＲを行ない、感染細胞内のＨＣＶを検出した。
【図８】それぞれのサンプルよりＲＮＡを抽出し、ＲＴ-ＰＣＲを行った後、アガロース電気泳動にかけ、エチジウムブロミドで染色した結果を示す図面で、アンチセンスオリゴヌクレオチドによるＨＣＶの複製阻害効果を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a recombinant hepatocyte producing HCV RNA polymerase, an HCV replication method comprising contacting HCV with the recombinant hepatocyte and culturing the same, and use thereof.
[0002]
[Prior art]
It has long been known that there are two types of viral hepatitis: hepatitis A (infectious hepatitis) and hepatitis B (serum hepatitis). This was mainly based on the difference in the route of infection, and it was confirmed that hepatitis A was epidemic by oral infection and hepatitis B was mainly transmitted via blood. As a definitive diagnostic method for these two hepatitis viruses has been established, the existence of non-A non-B hepatitis that does not belong to any of these has been revealed (Prince, AM, et al., Lancet I p.241, 1974).
[0003]
Among these, the genome of the hepatitis C virus, which is the main cause of transfusion-related non-A non-B hepatitis that infects via blood, was cloned and the entire nucleotide sequence was determined (Choo. QL, et al., Science 244, 359, 1989 and others). As a result, it was clarified that HCV is a virus closely related to flavi or pestiviruses with a genome of about 9.5 kb plus-strand RNA. However, hepatitis C has a viral concentration of 10 in patient serum.²-10^ThreeHowever, due to problems such as low infection and limited experimental models for chimpanzees and marmosets, the research has become difficult. For this reason, isolation and identification of the virus main body has not yet been made for hepatitis C.
[0004]
Under such circumstances, many laboratories have attempted to replicate HCV in an in vitro cell culture system. For this, primary hepatocytes are used as host cells (Ito. T., et al. J. Gen. Viol. 77, 1043-1054, 1996), and lymphocyte cells are used (Kato. N., Biochem. Biophys). Commun. 206, 863-869, 1995), those using mouse retrovirus-infected lymphocytes (Nakajima. N., J. Virol., 70, 3325-3329, 1996), cell lines derived from liver cancer (Tsuboi. S., J. Med. Virol., 48, 133-140, 1996) etc., and the establishment of infection has been reported. In addition, as a means to avoid the difficulty of constructing an in vitro culture system of HCV, there is a group that has constructed a chimeric poliovirus having a 5 ′ untranslated region of HCV and analyzed the function of the 5 ′ untranslated region of HCV (Lu. HH, Proc. Natl. Acad. Sci., 93, 1412-1417, 1996). However, the above method has problems such as a short virus replication period or a small amount of virus replication, and it has been difficult to obtain a sufficiently satisfactory result in studying inhibitors against HCV. Therefore, establishment of stable and easy HCV sensitive cells has been demanded.
[0005]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for efficiently and long-term replicating HCV by using recombinant hepatocytes that produce HCV RNA polymerase in an in vitro cell culture system.
[0006]
Another object of the present invention is to provide a method for detecting an HCV replication inhibitor using the above-described method for replicating HCV.
[0007]
[Means for Solving the Problems]
As a result of intensive studies to achieve the above object, the present inventors have succeeded in establishing a recombinant hepatocyte cell line that constitutively expresses HCV RNA polymerase. Furthermore, the inventors have found that HCV is sensitive to the recombinant hepatocytes, and that HCV can be replicated over a long period of time by continuing to culture the hepatocytes in contact with HCV, thereby completing the present invention. It was.
[0008]
The present invention also includes recombinant hepatocytes that produce HCV RNA polymerase and are sensitive to HCV.
[0009]
The present invention also includes a method for replicating HCV by contacting HCV with the above-described recombinant hepatocytes and culturing the recombinant hepatocytes for a long period of time.
[0010]
Furthermore, the present invention includes a method for detecting an HCV replication inhibitor by utilizing an HCV replication method. The present invention is described in further detail below.
[0011]
The recombinant hepatocytes and methods of the present invention are characterized by recombinant hepatocytes that produce HCV nonstructural proteins or RNA polymerase therein. The production of the recombinant hepatocytes is achieved by incorporating a DNA fragment encoding HCV nonstructural region protein or HCV RNA polymerase into an appropriate expression vector and introducing it into hepatocytes.
[0012]
DNA fragments encoding HCV non-structural region protein or HCV RNA polymerase are synthesized and amplified by RNA extraction, reverse transcriptase (RT) reaction and polymerase chain reaction (PCR) as described by Sambrook et al. According to a general method such as gene cloning (J. Sambrook, et. Al., Molecular Cloning. A laboratory manual. Cold Spring Harbor Laboratory, 1989), it is obtained from sera of patients with hepatitis C containing HCV. Preferably, a cDNA comprising the full length of the HCV genome constructed by ligating each fragment and inserting it into pBR322 based on the base sequence of HCV cDNA described in “JP-A-5-68563” of the published patent publication An integrated plasmid (designated pHCV-8) is used.
More specifically, the DNA fragment encoding the non-structural region protein of HCV is cleaved with pHCV-8 with an appropriate restriction enzyme such as EcoRV and EcoRI, and an appropriate linker such as a HindIII linker is added to the end. Then, each DNA fragment is separated by agarose electrophoresis, and it can be obtained by recovering an approximately 6.5 kb DNA fragment (referred to as 6.5 HCV) containing a gene encoding a nonstructural protein from the gel. In addition, a DNA fragment encoding HCV RNA polymerase was subjected to PCR using the synthetic primers described in SEQ ID NOs: 1 and 2 in the sequence listing, whereby the NS5B region containing the RNA polymerase gene (FIG. Obtained by cleaving with SalI and BamHI, separating each DNA fragment by agarose electrophoresis, and recovering a 1.77 kb DNA fragment (referred to as 1.77NS5B) from the gel it can. The thus obtained DNA fragment is incorporated into an appropriate expression vector.
[0013]
As an expression vector, a plasmid, a cosmid, a virus vector, or the like can be used. The promoter contained in the expression vector is selected from cytomegalovirus, early SV40, late SV40, β-actin and the like. If a product having RNA polymerase activity is produced, fusion with other proteins and peptides such as myc, β-galactosidase, glutathione-S-transferase, maltose binding protein, protein A, histidine hexamer, etc. It can also be expressed as a protein. Preferably, DNA fragment 6.5HCV is inserted into the HindIII cleavage site of expression plasmid pRc / CMV (Invitrogen), 1.77NS5B is HindIII- of expression plasmid pGST-E / CAG in which myc is previously inserted downstream of the promoter. Inserted into the SalI cleavage site. The expression plasmids thus obtained were named pRcHCNS6.5 and pGCNmycNS5B, respectively.
As marker genes, aminoglycoside 3 'phosphotransferase gene, dihydrofolate reductase gene, glutamine synthetase gene and the like can be used. Examples of these additive substances include G-418, neomycin, methotrexate and the like.
[0014]
For transformation of host cells, known methods such as calcium phosphate coprecipitation method, DEAE dextran method, lipofectin method, electroporation method and the like can be used, and an appropriate method may be selected depending on the host cell to be used. Preferably, lipofectin (GIBCO-BRL) is used, and hepatocytes are transformed according to the attached protocol. The host hepatocytes are suitably selected from human or chimpanzee primary hepatocytes, immortalized hepatocytes such as H8Ad17 and RY5, or established hepatocytes such as HepG2 and Alexander, and preferably HepG2. As the medium, a general synthetic medium that can be used for culturing hepatocytes is used.
[0015]
Recombinant hepatocytes have a sufficient time for the untransformed hepatocytes to die after 4 days of transformation, usually a few days to a few weeks, a medium commonly used, for example, a medium in which G418 is added to William E medium. It is obtained by culturing in. The thus obtained HCV RNA polymerase or the DNA fragment encoding the polymerase in the transformed hepatocytes is obtained by a commonly used method for detecting a protein or DNA fragment, such as Western blotting, Northern blotting, ELISA, RIA. Detected according to etc. More specifically, for detection of 6.5 HCV and 1.77 NS5B DNA fragments, mRNA was purified from recombinant hepatocytes using mRNA isolation kit (Boehringer), and RNA in the NS5B region labeled with digoxigenin ( Northern blotting using 366 bp) as a probe is performed. Further, HCV RNA polymerase in recombinant hepatocytes transformed with each plasmid can be indirectly detected by Western blotting. That is, as a primary antibody against recombinant hepatocytes transformed with pRcHCNS6.5, anti-NS5 rabbit IgG or anti-NS3 rabbit IgG is used as a primary antibody against recombinant hepatocytes transformed with pGCNmycNS5B. Alternatively, anti-NS5 rabbit IgG is used, respectively. It is also possible to use HCV positive human IgG as the primary antibody against both transformed hepatocytes.
[0016]
Whether or not HCV replicates in the above recombinant hepatocytes is determined by contacting the recombinant hepatocytes with serum containing HCV and then culturing them for a long period of time in the culture supernatant or cell lysate. It becomes clear by detecting HCV RNA or HCV antigen using a commonly used method such as in situ hybridization method, fluorescent antibody method, PCR method or ELISA method. More specifically, RNA in the sample is extracted according to the attached protocol using ISOGEN-LS (Nippon Gene) in the case of a culture supernatant and ISOGEN (Nippon Gene) in the case of cell sedimentation. Subsequently, the RNA is converted into cDNA with a reverse transcriptase such as MMLV (GIBCO), and then amplified by PCR, and the cDNA is detected by 1.5% agarose gel electrophoresis, whereby HCV Can be determined.
[0017]
The HCV-sensitive recombinant hepatocytes thus obtained can efficiently replicate HCV over a long period of time, and are HCV replication inhibitors, for example, oligonucleotides complementary to the HCV genome (also referred to as antisense oligonucleotides). A), and is used as a member of a screening system for neutralizing antibodies against HCV or other chemically synthesized substances capable of inhibiting HCV replication.
[0018]
【The invention's effect】
The present invention provides HCV-sensitive recombinant hepatocytes that constitutively produce HCV RNA polymerase and can efficiently replicate HCV over a long period of time.
[0019]
By using the above-described HCV-sensitive recombinant hepatocytes found by the present inventor, it is possible to provide a long-term efficient in vitro replication system for HCV, which has been considered difficult in the past, and use this HCV replication system As a result, it has become possible to develop therapeutic agents for HCV. In addition, since the HCV replication system of the present invention produces HCV or HCV-constituting proteins, the HCV-infected hepatocytes and HCV-infected hepatocyte lysate can be used for HCV antigen and antibody detection systems. It becomes a material for constructing diagnostic agents for infection.
EXAMPLES Hereinafter, although this invention is demonstrated further in detail along an Example, these Examples demonstrate various specific examples of this invention, and this invention is not limited to these Examples. Absent.
[0020]
【Example】
Example 1: Construction of an expression plasmid containing an RNA polymerase gene region
The pHCV-8 used in this example is based on the nucleotide sequence described in “JP-A-5-68563” of the published patent gazette and uses each clone of the λgt11 phage vector into which the HCV cDNA fragment described in the same report is inserted. It was constructed according to the method of Sambrook. That is, by excising the HCV cDNA fragment from each λgt11 vector by appropriate restriction enzyme treatment and agarose electrophoresis, and repeating the ligation of the HCV cDNA fragment and the insertion into pBR322, the entire length of the HCV genome was finally reduced to EcoRI and HindIII of pBR322. Plasmid pHCV-8 inserted at the site was constructed.
[0021]
Ten units of restriction enzyme EcoRV (Takara Shuzo) and 15 units of EcoRI (Takara Shuzo) were added to 2 μg of pHCV-8 and reacted at 37 ° C. for 2 hours. The reaction product was treated with phenol / chloroform and then precipitated with ethanol to recover the DNA fragment. This DNA fragment was dissolved in 30 μl of water, 4 units of T4 DNA polymerase (Takara Shuzo Co., Ltd.) and 3 μl of 2 mM dNTP mixture were added thereto, and reacted at 37 ° C. for 30 minutes to repair the cleavage ends generated by restriction enzymes. . This DNA fragment was recovered by the same method as described above and dissolved in 20 μl of water.
To the 10 μl of the solution, HindIII linker (Takara Shuzo) and DNA ligation kit (Takara Shuzo) were added, and a ligation reaction was performed according to the manufacturer's protocol. After completion of the reaction, DNA was recovered by the same method as described above, 15 units of HindIII was added thereto and reacted at 37 ° C. for 2 hours. This reaction solution was subjected to 0.8% agarose gel electrophoresis, and 6.5 kb 6.5 HCV containing the nonstructural gene region was extracted and collected from the gel. The 6.5 HCV was added to 100 ng of an expression vector pRc / CMV for animal cells that had been treated with HindIII, and a ligation reaction was performed using a DNA ligation kit. The expression plasmid constructed in this way was named pRcHCNS6.5 (FIG. 2).
[0022]
A plasmid expressing only the NS5B region containing the gene encoding HCV RNA polymerase was constructed as follows. First, the oligonucleotide according to SEQ ID NO: 3 in the sequence listing, in which a HindIII cleavage sequence is added to the 5 ′ end of the base sequence encoding a tag consisting of 12 amino acids to which 10 amino acids of myc have been added, is complementary to this Then, an oligonucleotide described in SEQ ID NO: 4 with a SalI cleavage sequence added to its 5 ′ end was synthesized (Sawaday), and these oligo DNAs were annealed. This oligo DNA was added to 100 ng of an expression vector pGST-E / CAG for animal cells which had been cleaved with HindIII and SalI, and a ligation reaction was performed using a DNA ligation kit. The plasmid thus obtained was named N-12 (FIG. 3).
[0023]
Next, primers shown in SEQ ID NOs: 1 and 2 were synthesized, and the NS5B region of pHCV-8 was amplified by PCR using these primers. Specifically, 100 μmol / μl of primer was used for each 1 μl, 10 × PCR buffer 10 μl, 25 mM MgCl.₂6 μl, 2.5 mM dNTP mix 8 μl, template DNA (pHCV-8) 100 ng, recombinant Taq DNA polymerase (5 u / μl) (Takara Shuzo) 0.5 μl were mixed, and water was added to make a total volume of 100 μl. This reaction solution was subjected to PCR amplification for 35 cycles with PC-800 (Astech). PCR was performed under the following conditions. After 95 ° C for 1 minute, 55 ° C for 2 minutes and 72 ° C for 2 minutes of reaction, 94 ° C for 1 minute, 55 ° C for 2 minutes and 72 ° C for 2 minutes for 33 cycles, followed by 94 ° C for 1 minute PCR amplification was performed for 2 cycles at 72 ° C. for 5 minutes, a total of 35 cycles. After completion of the reaction, the amplification product was recovered by phenol / chloroform treatment and subsequent ethanol precipitation. This was dissolved in 20 μl of water, 10 units of SalI (Takara Shuzo) and 10 units of BamHI (Takara Shuzo) were added and reacted at 37 ° C. for 2 hours. After the reaction, 1% agarose gel electrophoresis was performed to recover a 1.77 kb DNA fragment 1.77NS5B (FIG. 4). The 1.77NS5B thus obtained was added to 100 ng of the N-12 plasmid shown in FIG. 3 that had been treated with SalI and BamHI, and a ligation reaction was performed using a DNA ligation kit. The obtained expression plasmid was designated as pGCNmycNS5B (FIG. 5).
[0024]
Example 2: Introduction of expression plasmid containing 1.77NS5B into hepatocytes and acquisition of recombinant hepatocytes
The previously constructed expression plasmid pRcHCNS6.5 or pGCNmycNS5B was introduced into HepG2 hepatocytes (ATCC deposit number: HB-8065) using lipofectin (GIBCO-BRL). The introduction method followed the protocol attached to the manufacturer. Specifically, after preparing a DNA solution in which 1 μg of expression plasmid was added to 100 μl of Opti-MEM medium (GIBCO) and a solution in which 10 μl of 1 mg / ml lipofectin was added to 100 μl of Opti-MEM medium, respectively. Mix gently and let stand for 10-15 minutes. To 200 μl of this mixed solution, 800 μl of Opti-MEM medium was added, and the entire amount was added to 1 × 10 5 in a 35 mm culture dish.^FiveWas added to the HepG2 hepatocytes seeded in (1). The cells were treated at 37 ° C with CO₂After standing overnight in an incubator, the medium was replaced with William E medium containing 10% FBS, and the culture was further continued for 48 hours. Cells grown on one side of the culture dish were collected, diluted 10-fold with William E medium, and expanded to a 10 cm culture dish. On the next day, the selection marker G418 (Sigma: geneticin) was replaced with a William E selective medium added to a final concentration of 1 mg / ml, and the culture was continued. The medium was replaced with the above selective medium every 3-5 days, and G418-resistant cells were obtained, separated and expanded after 2-3 weeks.
[0025]
Example 3: Confirmation of expression of inserted gene product
G418-resistant cells were each isolated and expanded by 15 clones, and the expression derived from the inserted gene was analyzed at the RNA level and the protein level.
Analysis at the RNA level was by Northern blotting. That is, 25cm²The above recombinant hepatocytes that became confluent in the culture dish were collected and 10⁷MRNA was purified from corresponding cells using mRNA isolation Kit (Boehringer), subjected to 1% agarose electrophoresis according to a conventional method, and then transferred to HybondN + (Amersham). Subsequently, the target RNA was detected using a 366 bp RNA probe labeled with digoxigenin synthesized using the DNA fragment shown in SEQ ID NO: 5 in the sequence listing corresponding to the NS5B region of the HCV genome as a template. As a result, a signal specific to the recombinant cells was detected.
[0026]
Analysis at the protein level was by Western blotting. That is, 25cm²Recombinant hepatocytes are collected from the culture dish, treated with cell treatment buffer (5 mM TrisHCl, 5% SDS, 2.5% 2-mercaptoethanol, 12.5% glycerol, 0.01% BPB), and then the lane. 10 per hit^FiveWere run on a 10-20% gradient gel (Daiichi Kagaku). After electrophoresis at a constant current of 40 mA for 1 hour, the protein was transferred to a PVDF membrane (Millipore) with SEMI-DRY TRANSFER CELL (BIO-RAD), and a detection reaction was performed according to a conventional method. As the primary antibody, anti-NS5 rabbit IgG, anti-NS3 rabbit IgG, anti-myc mouse monoclonal antibody or HCV positive human IgG was used. The protein transferred onto the PVDF membrane was detected using a BM Chemiluminescence Western Blotting Kit (Boehringer) using chemiluminescence according to the attached protocol. As a result, a signal having an expected size specific to recombinant hepatocytes was detected (FIG. 6). The recombinant hepatocyte in which pRcHCNS6.5 thus obtained was incorporated and produced NS3 and NS5, which are HCV-constituting proteins, was named HepG2 Dec4. In addition, a recombinant hepatocyte in which pGCNmycNS5B was incorporated and expressed NS5B was named NmycNS5B-3.
[0027]
Example 4: HCV infection experiment (1)
Three plasmas 6684, 5269, and 7878 with high HCV infectious titer and full length HCV RNA detected by Northern blot analysis were mixed in equal amounts, and this was further doubled with serum-free William E medium. Diluted material was used as infectious material.
HepG2 Dec4 suspended in William E medium containing 10% FBS was added to a 24-well culture plate at 1 × 10 6 per well.^FiveIndividual seeds were seeded and adsorbed overnight. The next day, add 1 ml of the infectious material to each well and add 37 ° C CO²Adsorbed overnight in incubator. The next day, the supernatant was removed by aspiration and washed 3 times with PBS, and 1 ml of 10% FBS-containing William E medium was added to start the culture. Four days after inoculation, cells were expanded into 6-well culture plates and another 25 cm after 4 days.²Expanded to flask. Furthermore, 4 days later, only the medium was replaced with 2% or 10% FBS-containing William E medium, and the cells were passaged after another 3 days. This weekly medium exchange and cell passage were repeated, and each time the cells were passaged, the culture supernatant and cells were sampled for PCR assay.
[0028]
HCV RNA is detected by extracting RNA from a sample, synthesizing cDNA by RT reaction, amplifying HCV cDNA by PCR using synthetic primers described in SEQ ID NOs: 6 to 17 in the Sequence Listing, and then adding 1.5% agarose. Gel electrophoresis was performed to detect a cDNA fragment having the desired size. PCR was performed in two stages, an outer primer set and an inner primer set. Three primer regions were used, and their locations and sequences are shown in FIG. More specifically, first, 750 μl of ISOGEN-LS was added to 250 μl of culture supernatant at 2-4 × 10 4.⁶For each cell sedimentation, 1 ml of ISOGEN is added, and 20 μl of H is added to the RNA extracted according to the attached protocol.₂O was added to dissolve, heated at 65 ° C. for 5 minutes, and then rapidly cooled. This is H₂O 22.67 μl, 10 × reaction buffer 5 μl, ribonuclease inhibitor (GIBCO-BRL) 0.18 μl, each 25 mM dNTP mixed solution 0.4 μl, 50 μM primer set forth in SEQ ID NOs: 6 and 9 in the sequence listing for each 0 0.5 μl, MMLV 0.5 μl, ampli Taq (Perkin) 0.25 μl were added, the total amount was 50 μl, and RT reaction was performed at 42 ° C. for 30 minutes, followed by 35 cycles of PCR amplification with PC-800 (Astech). In order to perform the second stage PCR amplification, 2.5 μl of the first stage PCR product was collected,₂O 19.55 μl, 10 × reaction buffer 2.5 μl, 25 μm each of dNTP mixed solution 0.1 μl, 50 μM primer described in SEQ ID NO: 7 and 8 of Sequence Listing, 0.125 μl each, ampli Taq 0.1 μl added, 35 cycles of PCR amplification were performed with a total volume of 25 μl. 10 μl of the second-stage PCR product was collected and subjected to 1.5% agarose gel electrophoresis to determine HCV replication. The observation results for 99 days are shown in Table 1. ● represents HCV positive, and ◯ represents HCV negative. HCV was detected intermittently over 99 days.
[0029]
[Table 1]

[0030]
Example 5: HCV infection experiment (2)
HCV infection experiments were performed using NmycNS5B-3 recombinant hepatocytes. After adding the infectious material to the recombinant cells,₂All were carried out in the same manner as in Example 4 except that the adsorption was performed for 4 hours in the incubator. The observation course for 56 days is shown in Table 2. ● represents HCV positive, and ◯ represents HCV negative. As a result, HCV was detected over 56 days. In HepG2 cells into which NS5B gene was not introduced, HCV was not detected during the 28-day observation period.
[0031]
[Table 2]

[0032]
Example 6: In situ hybridization
Intracellular HCV RNA was detected by in situ hybridization.
Infected cells suspended in Williams E medium containing 10% FBS were transferred to each well of 4 well LabTek Chamber (Nunk) at 5 × 10 5.^FourIndividual seeds were seeded and adsorbed overnight at 37 ° C. The next day, the cells were fixed with paraformaldehyde, and then 0.2 μg / ml proteinase K was allowed to act for 5 minutes. A digoxigenin-labeled RNA probe was added to this sample, and hybridization was performed overnight at 55 ° C. A probe for detecting the HCV genome was prepared as follows. First, 359 bases from the 342nd base, which is the translation initiation codon of HCV, to the 700th base were amplified by PCR. At that time, a T7 RNA polymerase recognition sequence was added to the downstream primer. The HCV DNA described in SEQ ID NO: 8 in the sequence listing thus amplified was used as a template for RNA probe synthesis. The synthesis of the RNA probe was performed by the digoxigenin labeling method using T7 RNA polymerase (Boehringer) and reagents according to the attached protocol.
[0033]
After hybridization, 5 × SSC (20 × SSC diluted 4 times) at 55 ° C. 20 × SSC contains 175.3 g NaCl and 88.2 g sodium citrate in H₂The solution was adjusted to pH 7.0 with 1 liter of O), and washed twice for about 1 minute, 2 × SSC for 20 minutes, and then 0.2 × SSC for 20 minutes. After completion of the washing operation, alkaline phosphatase-labeled anti-digoxigenin antibody (Boehringer) was reacted at room temperature for 30 minutes. Next, BCIP 33 μl and NBT 44 μl were added and reacted at room temperature for 6 hours or overnight. After the reaction, the cells were stained with an acetocarmine solution, dehydrated, sealed, and observed with an optical microscope.
[0034]
Example 7: Fluorescent antibody method
Infected cells suspended in William E medium containing 10% FBS were transferred to each well of a 4 well Lab Tek Chamber (Nunk) 5 × 10^FourIndividual seeds were seeded and adsorbed overnight at 37 ° C. On the next day, cells were fixed with 80% cold ethanol, and then fluorescent antibody reaction was performed according to a conventional method. As the primary antibody, a monoclonal antibody prepared by immunizing a mouse with an antigen purified by expressing the core protein of the HCV genome in E. coli was used. The monoclonal antibody was subjected to a primary antibody reaction at a concentration of 10 μg / ml. As the secondary antibody, FITC-labeled anti-mouse IgG (Cappel) was used.
[0035]
Example 8: Inhibition of HCV replication by antisense oligonucleotides
NmycNS5B-3 cells suspended in Wilam E medium containing 10% FBS were placed in a 24-well plate at 5 × 10 5 per well.^FourSeeded and cultured at 37 ° C. The volume of the culture solution was 1 ml. Two days later, the cells were washed with PBS and replaced with William E medium containing 2% FBS. Next, an oligonucleotide (Gleiner Japan) having a phosphorothioate bond in the skeleton as shown in SEQ ID NO: 19 in the sequence listing and complementary to the base sequence of HCV, and having the same sequence, and adding cholesterol to 3 ′ The oligonucleotide was added so that the final concentration was 10 μM in the former case and 1 μM in the latter case, and cultured for 1 day. The same treatment was carried out using an oligonucleotide having a sequence unrelated to HCV as a negative target and the other components being the same. The next day cells were washed twice with PBS and infected with HCV. Infection with HCV was carried out by the method described in Example 4 except that the plasma of each of the infectious materials 5269 and 7878 was mixed in an equivalent amount and adsorbed at 37 ° C. for 2 hours. After the virus adsorption, the cells were washed 5 times with PBS, and 1 ml of William E medium containing 10% FBS was added to start the culture. Infected cells were treated again with oligonucleotide 3 days after inoculation. That is, the infected cells were washed once with PBS, replaced with 1 ml of William E medium containing 2% FBS, and oligonucleotides were added thereto so as to have the aforementioned concentrations. On the next day, 80 μl of FBS was added so that the final concentration was 10%, and the culture was continued. Further, the same nucleotide treatment operation was repeated 4 days later. Four days after the third oligonucleotide treatment, the culture supernatant and cells were collected, and HCV RNA was detected by the method described in Example 4.
[0036]
The result is shown in FIG. Numbers 1 and 2 in the figure show the results of normal human serum. 3 to 6 show the results of oligo-treated HCV-infected NmycNS5B-3 cells. 3 is treatment with the oligonucleotide of SEQ ID NO: 19, 4 is treatment with the negative target oligonucleotide, 5 is treatment with the oligonucleotide of SEQ ID NO: 19 added with cholesterol at the 3 ′ end, and 6 is cholesterol at the 3 ′ end. Each of the treatments with the added negative target oligonucleotide is shown. 7 shows HCV-infected NmycNS5B-3 cells not treated with oligonucleotide. 8 shows oligo-untreated HCV-infected HepG2 cells. 9 shows HCV non-infected NmycNS5B-3 cells. 10 shows the result of HCV positive plasma. When the oligonucleotide of SEQ ID NO: 19 was treated, an HCV replication inhibitory effect was observed.
[0037]
[Sequence Listing]
SEQ ID NO: 1
Sequence length: 30
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids (synthetic DNA)
Array

[0038]
SEQ ID NO: 2
Sequence length: 33
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids (synthetic DNA)
Array

[0039]
SEQ ID NO: 3
Sequence length: 42
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids (synthetic DNA)
Array

[0040]
SEQ ID NO: 4
Sequence length: 42
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids (synthetic DNA)
Array

[0041]
SEQ ID NO: 5
Sequence length: 366
Sequence type: Nucleic acid
Number of chains: 2 chains
Topology: Linear
Sequence type: cDNA to genomic RNA
Other information: Corresponds to positions 9041 to 9406 in the base sequence of HCV genome type 1b.
Array

[0042]
SEQ ID NO: 6
Sequence length: 20
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids (synthetic DNA)
Array

[0043]
SEQ ID NO: 7
Sequence length: 20
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids (synthetic DNA)
Array

[0044]
SEQ ID NO: 8
Sequence length: 20
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids (synthetic DNA)
Array

[0045]
SEQ ID NO: 9
Sequence length: 20
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids (synthetic DNA)
Array

[0046]
SEQ ID NO: 10
Sequence length: 21
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids (synthetic DNA)
Array

[0047]
SEQ ID NO: 11
Sequence length: 21
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids (synthetic DNA)
Array

[0048]
SEQ ID NO: 12
Sequence length: 21
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids (synthetic DNA)
Array

[0049]
SEQ ID NO: 13
Sequence length: 21
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids (synthetic DNA)
Array

[0050]
SEQ ID NO: 14
Sequence length: 20
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids (synthetic DNA)
Array

[0051]
SEQ ID NO: 15
Sequence length: 20
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids (synthetic DNA)
Array

[0052]
SEQ ID NO: 16
Sequence length: 20
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids (synthetic DNA)
Array

[0053]
SEQ ID NO: 17
Sequence length: 20
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids (synthetic DNA)
Array

[0054]
SEQ ID NO: 18
Sequence length: 359
Sequence type: Nucleic acid
Number of chains: 2 chains
Topology: Linear
Sequence type: cDNA to genomic RNA
Other information: Corresponds to positions 342 to 700 of the base sequence of HCV genome type 1b.
Array

[0055]
SEQ ID NO: 19
Sequence length: 20
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: Other nucleic acids (synthetic DNA)
Other information: It corresponds to the 340th to 359th base sequence of the HCV genome type 1b, and is complementary to this, and has a phosphorothioate bond in the skeleton.
Array

[Brief description of the drawings]
FIG. 1 shows the gene structure of the HCV genome.
FIG. 2 shows a schematic diagram of the construction of an expression plasmid pRcHCNS6.5 in which a DNA fragment (6.5HCV) containing a gene region encoding an HCV nonstructural protein is inserted into the expression vector pRc / CMV.
FIG. 3 shows a schematic diagram of the construction of an expression plasmid N-12 in which a myc tag is inserted into the expression vector pGST-E / CAG. An oligonucleotide encoding 12 amino acids including 10 amino acids of myc and its complementary strand were synthesized, annealed, and inserted downstream of the promoter of the expression vector.
FIG. 4 is a schematic view for preparing a DNA fragment (1.77NS5B) containing a gene encoding HCV RNA polymerase.
5 shows a schematic diagram of the construction of an expression plasmid pGCNmycNS5B in which 1.77NS5B of FIG. 4 is inserted downstream of the N-12 myc tag of FIG.
FIG. 6 is a schematic diagram showing the results obtained by subjecting hepatocytes HepG2Dec4 introduced with pRcHCNS6.5 and NmycNS5B-3 introduced with pGCNmycNS5B to SDS-PAGE and identifying the expression of a protein derived from the HCV gene by Western blotting. .
FIG. 7 shows the positions of primers used for RT-PCR on the HCV genome. RT-PCR was performed using region A, region B or region C to detect HCV in the infected cells.
FIG. 8 is a drawing showing the results of extracting RNA from each sample, performing RT-PCR, subjecting it to agarose electrophoresis and staining with ethidium bromide, and showing the inhibitory effect of antisense oligonucleotide on HCV replication.

Claims (5)

        HCV replication characterized by contacting HCV with a recombinant hepatocyte producing the RNA polymerase, into which an RNA polymerase gene derived from hepatitis C virus (HCV) is incorporated, and culturing the recombinant hepatocyte Method.         The replication method according to claim 1, wherein the hepatocytes are derived from human.         The replication method according to claim 2, wherein the hepatocytes are HepG2.         A method for detecting an HCV replication inhibitor, which uses the replication method according to any one of claims 1 to 3.         The detection method according to claim 4, wherein the HCV replication inhibitor is selected from antisense nucleic acid substances, antibodies, and chemically synthesized substances.

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