JP2007289204A - Construct and method for expression of recombinant hcv envelop protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a leader peptide capable of using for production of HCV envelop protein as well as HCV envelop protein obtained thereby. <P>SOLUTION: Disclosed are recombinant nucleic acids comprising an avian lysozyme leader peptide linked to HCV envelop protein or one part thereof, or a nucleotide sequence encoding a protein including a functional equivalent thereof, and HCV envelop protein obtained thereby. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to the general field of recombinant protein expression. More particularly, the present invention relates to hepatitis C virus envelope proteins in eukaryotes such as yeast. Constructs and methods for the expression of yeast core glycosylated viral envelope proteins are disclosed.

BACKGROUND OF THE INVENTION Hepatitis C virus (HCV) infection is a major health problem in both developing and developing countries. It is estimated that about 1-5% of the world population is affected by this virus. HCV infection is believed to be the most important cause of hepatitis from blood transfusions and often progresses to chronic liver damage. Moreover, there is evidence that HCV is involved in the induction of hepatocellular carcinoma. As a result, there is a high demand for reliable diagnostic methods and effective therapeutic agents. Similarly, there is a need for specific screening methods that are sensitive to HCV-infected blood products and improved methods for culturing HCV.

  HCV is a positive strand RNA virus of about 9600 bases that encodes a single polyprotein precursor of about 3000 amino acids. Proteolytic cleavage of this precursor combined with post-translational and co-modification has been shown to result in at least 3 structural proteins and 6 nonstructural proteins. Based on sequence homology, structural proteins have been functionally assigned as one single core protein and two envelope glycoproteins, E1 and E2. The E1 protein consists of 192 amino acids and contains 5-6 N-glycosylation sites, depending on the HCV genotype. The E2 protein is composed of 363-370 amino acids and contains 9-11 N-glycosylation sites depending on the HCV genotype (see Non-Patent Documents 1 and 2 for review). ). The E1 protein contains various variable domains (Non-Patent Document 2), while the E2 protein contains three hypervariable domains, of which the main domain is at the N-terminus of the protein (Non-Patent Document 2). HCV glycoproteins are mostly localized within the ER, where these proteins are modified and assembled into an oligomer complex.

  In eukaryotes, sugar residues are generally linked to four different amino acid residues. These amino acid residues are classified as O-linked (serine, threonine and hydroxylysine) and N-linked (asparagine). O-linked sugars are synthesized from nucleotide sugars in rough endoplasmic reticulum (ER) or Golgi. N-linked sugars are synthesized from a common precursor and then processed. HCV envelope proteins are thought to be N-glycosylated. In the art, N-linked carbohydrate chains are used for stabilization of folding intermediates and thus efficient folding, prevention of folding and degradation in the endoplasmic reticulum, oligomerization, biological activity and transport of sugar proteins. It is known that addition is important (see Non-Patent Documents 3 to 5). The tripeptide sequences Asn-X-Ser and Asn-X-Thr (where X can be any amino acid) on the polypeptide are consensus sites for binding N-linked oligosaccharides. After addition of an N-linked oligosaccharide to the polypeptide, the oligosaccharide is further complex type (containing N-acetylglucosamine, mannose, fucose, galactose and sialic acid) or containing N-acetylglucosamine and mannose. Processed to high mannose type. HCV envelope proteins are believed to be of high mannose type. N-linked oligosaccharide biosynthesis in yeast is very different from that in mammals. In yeast, oligosaccharide chains are elongated in the Golgi through the stepwise addition of mannose, leading to a sophisticated high mannose structure that does not contain sialic acid. In contrast, proteins expressed in prokaryotes are never glycosylated.

  To date, vaccination against disease has proven to be the most cost-effective and effective way to control disease. However, despite promising results, research efforts to develop effective HCV vaccines have been challenging. A prerequisite for a vaccine is the induction of an immune response in the patient's body. Therefore, HCV antigenic determinants must be identified and administered to the patient in an appropriate environment. Antigenic determinants can be divided into at least two forms: linear and conformational epitopes. Conformational epitopes are the result of molecular folding in three-dimensional space, including simultaneous translation and post-translational modifications such as glycosylation. In general, conformational epitopes represent epitopes that are similar to native-like HCV epitopes and may be better preserved than the actual linear amino acid sequence, so these epitopes are the most effective vaccines. It is thought that will be realized. Thus, the degree of accidental glycosylation of the HCV epitope protein is of greatest importance for generating a native-like HCV antigenic determinant. However, it appears that there are problems that cannot be overcome in culturing HCV that results in only a small amount of virions. Furthermore, there are enormous problems associated with the expression and purification of recombinant proteins that result in either small amounts of protein, either hyperglycosylated or non-glycosylated proteins.

  In order to obtain glycosylation of the expressed protein, the protein needs to be targeted to the endoplasmic reticulum (ER). This process requires the presence of a pre- or prepro sequence, also known as a signal peptide or leader peptide, at the amino terminus of the expressed protein. At the time of translocation of the protein into the lumen of the ER, the presequence is removed using a signal peptidase complex. A large number of prepros and prosequences are currently known in the art. These include S.I. cerevisiae a-mating factor leader (prepro; αMF or MFα), Carcinus mainas hyperglycemic hormone leader sequence (pre; CHH), S. cerevisiae. Occidentalis amylase leader sequence (pre; Amy1), S. occidentalis glucoamylase Gam1 leader sequence (pre; Gam1), fungal phytase leader sequence (pre; Phy5), Pichia pastoris acid phosphatase leader sequence (pre; Pho1), yeast aspartic protease 3 signal peptide (pre; YAP3), mouse salivary amylase A signal peptide (pre) and a chicken lysozyme leader sequence (pre; CL) are included.

  The CHH leader is coupled with hirudin and G-CSF (granular colony stimulating factor) and the expression of CHH-hirudin and CHH-G-CSF protein in Hansenula polymorpha results in proper removal of the leader sequence. (Non-patent Document 6 and Patent Document 1). The chicken lysozyme leader sequence is fused to human interferon α2b (IFNα2b); human serum albumin and human lysozyme or 1,4-β-N-acetylmuramidase. It was expressed in C. cerevisiae (Rapp accession number AF405538 in GenBank, Non-Patent Document 7 and Patent Documents 2 and 3). Mustilli and co-workers (Non-Patent Document 8) cerevisiae and K. et al. A Kluyveromyces lactis killer toxin leader peptide was utilized for the expression of HCV E2 in lactis.

  HCV envelope proteins have been produced by recombinant techniques in Escherichia coli, insect cells, and yeast cells. However, expression in higher eukaryotes has been characterized by the problem of obtaining large amounts of antigen to produce vaccines in some cases. E. Expression in prokaryotes such as E. coli results in a non-glycosylated HCV envelope protein. Expression of HCV envelope protein in yeast resulted in hyperglycosylation. As already demonstrated in U.S. Patent No. 6,047,033, expression of HCV envelope protein E2 in Saccharomyces cerevisiae leads to a highly glycosylated protein. This hyperglycosylation leads to shielding of protein epitopes. (Non-Patent Document 8) While asserting that the expression of HCV E2 in C. cerevisiae results in core-glycosylation, the results of the intracellularly expressed material demonstrate that a portion of it is at least hyperglycosylated. However, proper processing of the rest of the material was not shown. The need for HCV envelope proteins derived from intracellular sources is well accepted (Patent Document 4). This need is derived from chimpanzee sera immunized with mammalian cell culture-derived E2 protein and secreted yeast, as demonstrated in FIG. 5 of Mustilli and co-workers (Non-Patent Document 8). This is illustrated by the low reactivity with E2. This is further substantiated by Rosa (non-patent document 9) and co-workers who have shown that immunization with yeast-derived HCV envelope proteins cannot provide protection against antigen challenge.

Accordingly, there is a need for an efficient expression system that results in large amounts and cost-effective proteins, and such a system is required for the production of HCV envelope proteins. If a pre- or pre-pro sequence is used to direct the protein of interest to the ER, the efficiency of the expression system is inter alia due to the efficiency and fidelity with which the pre- or pre-pro sequence is removed from the protein of interest. It depends.
International Publication No. WO00 / 40727 European Patent No. EP0362183 European Patent No. EP0184575 International Publication No. WO96 / 04385 Major, M.M. E. And Feinstone, S .; M.M. 1997 Maertens, G.M. And Stuyver, L .; 1997 Rose, J. et al. K. And Doms, R .; W. 1988 Doms, R.D. W. Et al., 1993 Helenius, A.M. 1994 Weydemann, U. Et al., 1995 Okabayashi, K .; Et al., 1991 Mustilli, A.M. C. Et al., 1999 Rosa, D.C. Et al., 1996

  An object of this invention is to provide the leader peptide which can be used for production of HCV envelope protein, and the HCV envelope protein obtained by using it.

SUMMARY OF THE INVENTION A first aspect of the invention is a recombinant nucleic acid comprising a nucleotide sequence encoding a protein comprising an avian lysozyme leader peptide or a functional equivalent thereof bound to an HCV envelope protein or a portion thereof. About. More specifically, the protein is characterized by the structure CL-[(A1) _a- (PS1) _b- (A2) _c ] -HCVENV-[(A3) _d- (PS2) _e- (A4) _f ]. age,
In the formula;
CL is an avian lysozyme leader peptide or a functional equivalent thereof;
A1, A2, A3 and A4 are adapter peptides that can be different or the same;
PS1 and PS2 are processing sites that can be different or the same;
HCVENV is an HCV envelope protein or part thereof;
a, b, c, d, e and f are 0 or 1,
Here, A1 and / or A2 may be a part of PS1, and / or A3 and / or A4 may be a part of PS2.

  The recombinant nucleic acid according to the present invention may further comprise a regulatory element that allows expression of said protein in a eukaryotic host cell.

  Another aspect of the present invention relates to a recombinant nucleic acid according to the present invention contained in a vector. The vector may be an expression vector and / or an autonomously replicating vector or an integrating vector.

A further aspect of the invention relates to a host cell comprising a recombinant nucleic acid according to the invention or a vector according to the invention. More particularly, the host cell is capable of expressing a protein comprising an avian lysozyme leader peptide or a functional equivalent thereof bound to an HCV envelope protein or a portion thereof. More specifically, the protein is CL-[(A1) _a- (PS1) _b- (A2) _c ] -HCVENV-[(A3) _d- (PS2) _e- (A4) _f ].
Characterized by the structure;
In the formula;
CL is an avian lysozyme leader peptide or a functional equivalent thereof;
A1, A2, A3 and A4 are adapter peptides that can be different or the same;
PS1 and PS2 are processing sites that can be different or the same;
HCVENV is an HCV envelope protein or part thereof;
a, b, c, d, e and f are 0 or 1,
Here, A1 and / or A2 may be a part of PS1, and / or A3 and / or A4 may be a part of PS2.

  The host cells of the present invention can have the ability to remove avian lysozyme leader peptides with high efficiency and high fidelity, and process the processing sites PS1 and / or PS2 in the protein translocated to the endoplasmic reticulum Can have ability. The host cell may further have the ability to N-glycosylate the protein translocated to the endoplasmic reticulum or the protein translocated to the endoplasmic reticulum and processed at the sites PS1 and / or PS2. The host cell can be a eukaryotic cell such as a yeast cell.

In a next aspect of the present invention, there is provided a method for producing an HCV envelope protein or a part thereof in a host cell comprising the step of transforming said host cell with a recombinant nucleic acid of the present invention or a vector according to the present invention. A method comprising, wherein the host cell is capable of expressing a protein comprising an avian lysozyme leader peptide or a functional equivalent thereof bound to an HCV envelope protein or a portion thereof. More particularly, the host cell is: CL - a _{[(A4) f (A3)} d - - - (PS2) e] that the structure _{[(A1) a - (PS1)} b - (A2) c ] -HCVENV As a feature,
In the formula;
CL is an avian lysozyme leader peptide or a functional equivalent thereof;
A1, A2, A3 and A4 are adapter peptides that can be different or the same;
PS1 and PS2 are processing sites that can be different or the same;
HCVENV is an HCV envelope protein or part thereof;
a, b, c, d, e and f are 0 or 1,
Here, A1 and / or A2 may be a part of PS1, and / or A3 and / or A4 may be a part of PS2.

  The method of the invention further comprises culturing the host cell in an appropriate medium to obtain expression of the protein, isolating the expressed protein from or from the culture of the host cell. Also good. Said isolation comprises (i) lysis of said host cell in the presence of a chaotropic agent; (ii) chemical modification of the thiol group of cysteine in the isolated protein, which is reversible or irreversible; and (Iii) One or more of heparin affinity chromatography may be included.

  The present invention provides a recombinant nucleic acid encoding HCV envelope protein and avian lysozyme leader peptide, a method for producing HCV envelope protein, and the like.

Detailed Description of the Invention In the research work leading to the present invention, expression of HCV envelope protein as αMF-HCVENV (α mating factor-HCV envelope protein) in Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha is possible, It has been observed that the degree of pre-pro or pre-sequence removal is unacceptably low, and very often pre-pro or pre-sequence removal does not occur with high fidelity. As a result, a number of different HCV envelope proteins are produced in these yeasts that do not have a natural amino terminus (see Example 15). Most of the HCV envelope proteins expressed in these yeast species were glycosylated (see Examples 6, 10, 13 and 25). More particularly, cerevisiae (glycosylation-deficient mutant) and H. cerevisiae. The HCV envelope protein expressed by polymorpha was glycosylated in a manner similar to core glycosylation. The HCV envelope protein expressed in Pichia pastoris is also related to previous reports that the protein expressed in this yeast is not normally hyperglycosylated (Gellissen et al., 2000, Sugrue, RJ et al., 1997). Rather, it was hyperglycosylated.

  Constructs are made for expression of HCV envelope proteins as pre-pro or preprotein, where these prepros or presequences are Carcinus maenas hyperglycemic hormone leader sequence (pre; CHH), S. cerevisiae. Occidentalis amylase leader sequence (pre; Amy1), S. occidentalis glucoamylase Gam1 leader sequence (pre; Gam1), fungal phytase leader sequence (pre; Phy5), Pichia pastoris acid phosphatase leader sequence (pre; Pho1), yeast aspartic protease 3 signal peptide (pre; YAP3), mouse salivary amylase Either the signal peptide (pre) and the chicken lysozyme leader sequence (pre; CL. Only one of these prepro-HCVENV or pre-HCVENV proteins was pre-pro or with high frequency and high fidelity or The removal of the pre-sequence was observed, which was surprisingly found for the chicken lysozyme leader sequence (CL), S. cerevisiae and H. polymorpha. (See Example 16) The CL signal peptide thus shows very good performance for the expression of glycosylated HCV envelope proteins in eukaryotic cells. The findings are reflected in different aspects and embodiments of the invention as presented below.

  A first aspect of the present invention relates to a recombinant nucleic acid comprising a nucleotide sequence encoding a protein comprising an avian lysozyme leader peptide or a functional equivalent thereof bound to an HCV envelope protein or a portion thereof.

In its first embodiment, the recombinant nucleic acid comprising a nucleotide sequence is:
CL-[(A1) _a- (PS1) _b- (A2) _c ] -HCVENV-[(A3) _d- (PS2) _e- (A4) _f ]
It is characterized by the structure
In the formula;
CL is an avian lysozyme leader peptide or a functional equivalent thereof;
A1, A2, A3 and A4 are adapter peptides that can be different or the same;
PS1 and PS2 are processing sites that can be different or the same;
HCVENV is an HCV envelope protein or part thereof;
a, b, c, d, e and f are 0 or 1,
Here, A1 and / or A2 encodes a protein that may be part of PS1 and / or A3 and / or A4 may be part of PS2.

In a further embodiment, the recombinant nucleic acid of the invention comprises a protein or CL-[(A1) _a − comprising an avian lysozyme leader peptide or a functional equivalent thereof linked to an HCV envelope protein or a portion thereof. (PS1) _b- (A2) _c ] -HCVENV-[(A3) _d- (PS2) _e- (A4) _f ] allows expression of the protein in eukaryotic host cells characterized by the structure It further comprises an adjustment element.

  The terms “polynucleotide”, “polynucleic acid”, “nucleic acid sequence”, “nucleotide sequence”, “nucleic acid molecule”, “oligonucleotide”, “probe”, or “primer” as used herein. Means nucleotides, ie ribonucleotides, deoxyribonucleotides, peptide nucleotides or lock nucleotides, or combinations thereof, in any length or any form of polymeric form (eg, branched DNA). The term further includes double-stranded (ds) and single-stranded polynucleotides. The terms also include one or more naturally occurring nucleotide modifications such as methylation, cyclization and “caps” and analogs such as inosine or non-amplifiable monomers such as HEG (hexaethylene glycol). Also encompassed are nucleotide substitutions that occur. Ribonucleotides are denoted as NTP, deoxyribonucleotides as dNTP, and nudideoxyribonucleotides as ddNTP.

  Nucleotides can generally be labeled by radiation, chemiluminescence, fluorescence, phosphorescence or with infrared dyes or with surface enhanced Raman labels or plasmon resonance particles (PRP).

  The terms “polynucleotide”, “polynucleic acid”, “nucleic acid sequence”, “nucleotide sequence”, “nucleic acid molecule”, “oligonucleotide”, “probe”, or “primer” are similarly used when the backbone is not a sugar. Rather, it also includes peptide nucleic acids (PNA) that are DNA analogs that are pseudopeptides composed of N- (2-aminoethyl) -glycine units. PNA mimics the behavior of DNA and binds complementary nucleic acid strands. The neutral backbone of PNA results in stronger binding and greater specificity than is normally achieved. In addition, the unique chemical, physical and biological properties of PNA have been developed and used to produce powerful biomolecular means, antisense and antigenic agents, molecular probes and biosensors. PNA probes can generally be shorter than DNA probes, generally 6-20, more optimally 12-18 bases long (Nielsen, PE 2001) long It has. The term further encompasses locked nucleic acids, which are RNA derivatives in which the ribose ring is constrained by a methylene bond between the 2 'oxygen and the 4' carbon. LNAs exhibit unique binding affinity towards DNA or RNA target sequences. LNA nucleotides can be oligomerized and incorporated into chimeric or mixed-mer LNA / DNA or LNA / RNA molecules. LNA does not appear to be toxic to cultured cells (Orum, H. and Wengel, J. 2001, Wahistedt, C. et al., 2000). In general, any chimera or mixmer of DNA, RNA, PNA and LNA is considered as well as any of these in which thymine is replaced with uracil.

  The term “protein” refers to a polymer of amino acids and not a specific length of the product. Thus, peptides, oligopeptides and polypeptides are included within the definition of protein. The term likewise does not mean or exclude post-expression modifications of the protein, such as glycosylation, acetylation, phosphorylation and the like. Included within this definition are polypeptides that contain one or more analogs of amino acids (including, for example, unnatural amino acids, PNA, etc.), substituted bonds, and both naturally occurring and non-naturally occurring Of polypeptides with other modifications known in the art.

  As used herein, the term “preproprotein” or “preprotein” refers to a protein comprising a prepro sequence bound to a protein of interest or a protein comprising a pro sequence bound to the protein of interest. Each means. As an alternative to “pre-sequence”, terms such as “signal sequence”, “signal peptide”, “leader peptide” or “leader sequence” are used. All mean amino acid sequences that target the preprotein to the rough endoplasmic reticulum (ER), which is a prerequisite for (N-) glycosylation. A “signal sequence”, “signal peptide”, “leader peptide”, or “leader sequence” is a signal sequence bound to the protein of interest on the luminal side of this ER by a host-specific protease called a signal peptidase. Cleaved or “removed” from the containing protein. Similarly, preproproteins are converted to proproteins at the time of translocation to the lumen of the ER. Depending on the content of the “pro” amino acid sequence, it may or may not be removed by the host cell expressing the preproprotein. The well-known prepro amino acid sequence is S. cerevisiae. cerevisiae alpha mating factor alpha mating factor prepro sequence.

  “Recombinant nucleic acid” refers to at least one recombination, such as restriction enzyme digestion, PCR, ligation, dephosphorylation, phosphorylation, mutagenesis, codon adaptation for expression in heterologous cells, etc. A natural or synthetic nucleic acid that has been subjected to type DNA technology manipulation. In general, a recombinant nucleic acid is a fragment of a naturally occurring nucleic acid, otherwise comprising at least two nucleic acid fragments that are not naturally associated, or a fully synthetic nucleic acid.

The term “avian lysozyme leader peptide or functional equivalent thereof bound to an HCV envelope protein or a portion thereof” means that the C-terminal amino acid of the leader peptide is linked via a peptide bond to the N-terminal amino acid of the HCV envelope protein or a portion thereof. Means that they are covalently linked. Alternatively, the C-terminal amino acid of the leader peptide is separated from the N-terminal amino acid of the HCV envelope protein or a portion thereof by a peptide or protein. The peptide or protein may have a structure of-[(A1) _a- (PS1) _b- (A2) _c ] as described above.

The HCV envelope protein of interest or the structure CL-[(A1) _a- (PS1) _b- (AS) _c ] -HCVENV-[(A3) _d- (PS2) _e- (A4) _f ] may be induced in vivo by the proteolytic mechanism of the cell in which the protein is expressed. it can. More particularly, the step consisting of removal of the avian lysozyme leader peptide is preferably performed in vivo by the proteolytic mechanism of the cell in which the preprotein is expressed internally. However, induction is performed only after and / or during the isolation and / or purification of the preprotein and / or protein from cells in which the preprotein and / or cells expressing the preprotein are grown. You can also Alternatively, the in vivo induction is performed in combination with the in vitro induction. Induction of the HCV protein in question from the recombinantly expressed preprotein further degrades all or most of the contaminating proteins present at the same time as the protein of interest, and the protein in question is an abrasive proteolytic enzyme (multiple proteins). May include the use of one or more proteolytic enzymes in a polishing step that is resistant to Guiding and polishing are not mutually exclusive processes and can be obtained by using the same single proteolytic enzyme. As an example, the HCV genotype 1b HCVE1 protein lacking the Lys residue (SEQ ID NO: 2) is shown here. By digesting the protein extract containing the E1 protein with endoproteinase Lys-C (endo-lysC), the E1 protein is not degraded, while the contaminating protein containing one or more Lys residues is degraded. The Such a process can greatly simplify or enhance the isolation and / or purification of HCV E1 protein. Furthermore, inclusion of an additional Lys residue, for example between the leader peptide and the HCV E1 protein, within the preprotein provides the additional advantageous possibility of proper in vitro separation of the leader peptide from the HCV E1 preprotein. . Other HCV E1 proteins may be present at any one or more of positions 4, 40, 42, 44, 61, 65 or 179 (where position 1 is the first N-terminal natural amino acid of the E1 protein, ie HCV poly It can contain a Lys residue (position 192 in the protein). As mentioned above, the Lys residue can be mutated into another amino acid residue, preferably an Arg residue, to allow the use of endo-lysC.

  The term “properly removed” leader peptide means that the leader peptide has been efficiently removed from a protein containing a signal sequence attached to the protein of interest (ie, a large number of pre (pro) proteins are proproteins or Means converted to protein) and removed with high fidelity (ie, only the pre-amino acid sequence is removed and none of the amino acids of the protein in question attached to said pre-amino acid sequence are removed). “Efficient leader peptide removal” refers to at least about 40% of the preprotein, but more preferably about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 95%, 96%, 97%, 98% or even 99% means that it is converted into a protein from which the pre-sequence has been removed. Alternatively, if a substantial portion of the expressed preprotein is not converted to a protein from which the presequence has been removed, these preproteins can still be purified.

  “A functional equivalent of an avian lysozyme (CL) leader peptide” refers to a CL leader peptide in which one or more amino acids are replaced with another amino acid, so that this substitution is a conservative amino acid substitution Means. “Conservative amino acid substitution” means replacing one amino acid belonging to one conserved amino acid group with another amino acid belonging to the same conserved amino acid group. Conserved amino acid groups include: a group consisting of Met, Ile, Leu and Val; a group consisting of Arg, Lys and His; a group consisting of Phe, Trp and Tyr; a group consisting of Asn and Gln; a Cys, Ser and Thr. A group consisting of; and a group consisting of Ala and Gly are considered. Examples of conservative amino acid substitutions in the CL leader peptide include a natural mutation at position 6 and the amino acid at this position is Val or Ile; another variation occurs at position 17 and the amino acid at this position is among others , Leu or Pro (see SEQ ID NO: 1). Thus, the resulting CL leader peptide should be considered a functional equivalent. Other functional equivalents of CL leader peptides include leader peptides that reproduce the same technical aspects as CL leader peptides as described throughout the present invention, including deletion mutants and insertion mutants. It is.

“A” or “adapter peptide” means serving as a linker between, for example, a leader peptide and a processing site (PS), a leader peptide and a protein of interest, PS and a protein of interest, and / or a protein of interest and PS Means and / or a peptide (eg 1-30 amino acids) or a protein that can and / or serve as the linker N- or C-terminus of, for example, a leader peptide, PS or protein of interest. The adapter peptide “A” can have a certain three-dimensional structure such as, for example, an α-helix or β-sheet structure or a combination thereof. Alternatively, the three-dimensional structure of A is not clear and is, for example, a high-order coil structure. Adapter A can be, for example, a pre-sequence, a pro-sequence, a protein of interest sequence or part of a processing site. Adapter A can serve as a tag that allows or enhances the detection and / or purification and / or processing of the protein of which A is a part. An example of an A peptide is a his-tag peptide (HHHHHH: SEQ ID NO: 63) H _n where n is usually 6, but can be 7, 8, 9, 10, 11 or 12. Other examples of A-peptides, when present at the N-terminus of the protein of interest, not only increase fermentation yield but also protect the N-terminus of the protein of interest against processing by dipeptidylaminopeptidase, thus Included are peptides EEGEPK (Kjelsen et al .; in WO 98/28429; SEQ ID NO: 64) or EEAEPK (Kjelsen et al .; in WO 97/22706; SEQ ID NO: 65) reported as resulting in a homogeneous N-terminus of the peptide . At the same time, in vitro maturation of the protein of interest, ie removal of the peptides EEEEPK (SEQ ID NO: 64) and EEAEPK (SEQ ID NO: 65) from the protein of interest cleaves the C-terminus of the Lys residue within the peptide. This can be achieved by using lys C. Thus, the peptide functions as an adapter peptide (A) as well as a processing site (PS) (see below). The adapter peptides are given in SEQ ID NOs: 63-65, 70-72 and 74-82. Another example of an adapter peptide is a G4S immune silent linker. Other examples of adapter peptides or adapter proteins are listed in Table 2 of Stevens) (Stevens et al., 2000).

  “PS” or “processing site” refers to a site during or capable of processing a specific protein. Examples of processing sites that are susceptible to specific enzyme processing include IEGR ↓ X (SEQ ID NO: 66), IDGR ↓ X (SEQ ID NO: 67), AEGR ↓ X (SEQ ID NO: 68), all of which are bovine factor Xa Recognized by and cleaved between Arg and Xaa (any amino acid) residues represented by “↓” by the protease (Nagai, K. and Thogensen, H, C. 1984). Another example of a PS site is a dibasic site such as Arg-Arg, Lys-Lys, Arg-Lys, or Lys-Arg that can be cleaved by yeast Kex2 protease (Julius, D. et al. 1984). The PS site can also be a single base Lys site. The single base Lys-PS-site may be included at the C-terminus of the A peptide. Examples of A adapter peptides containing a C-terminal single base Lys-PS site are shown by SEQ ID NOs: 64-65 and 74-76. Exoproteolytic removal of His-tag (HHHHHH, SEQ ID NO: 63) uses dipeptidyl aminopeptidase I (DAPase) alone or in combination with glutamine cyclotransferase (Qcyclase) and pyroglutamine aminopeptidase (pGAPase) (Pedersen, J. et al. 1999). Said exopeptidase containing a recombinant His-tag (which allows removal of the peptidase from the reaction mixture by immobilized metal affinity chromatography, IMAC) is, for example, Unizyme Laboratories (Holsholm, DK). )) TAGZyme system. The term “processing” generally refers to any method or procedure that allows deproteinization to be specifically or cleavable at at least one processing site when said processing site is present in the protein. ing. PS may be susceptible to endo-type protease cleavage or may be susceptible to exo-type protease cleavage, but in any case, cleavage is specific. That is, it does not expand to a site other than the site recognized by processing the proteolytic enzyme. A certain number of PS sites are given in SEQ ID NOs: 66-68 and 83-84.

The versatility of the [(A1 / 3) _{a / d-} (PS1 / 2) _{b / e-} (A2 / 4) _{c / f} ] structure outlined above is demonstrated using several examples. The In the first example, the structure is present at the C-terminus of the protein of interest contained within the preprotein, where A3 is superimposed with the factor Xa “IEGRRX” PS site (SEQ ID NO: 66). Is the “VIEGR” peptide (SEQ ID NO: 69), where X = A4 is a histidine tag (SEQ ID NO: 63) (thus d, e and f are all 1 in this case). The HCV protein in question can be purified by () IMAC. After processing with factor Xa, the HCV protein of interest (optionally purified) will carry a processed PS site (SEQ ID NO: 70) that is “IEGR” at its C-terminus. Another processing site for the processed factor Xa can be IDGR (SEQ ID NO: 71) or AEGR (SEQ ID NO: 72). In a further example, the [(A1 / 3) _{a / d-} (PS1 / 2) _{b / e-} (A2 / 4) _{c / f} ]] structure is present at the N-terminus of the HCV protein in question. Furthermore, A1 is a histidine tag (SEQ ID NO: 63) and PS is a factor Xa recognition site (any of SEQ ID NOs: 66-68), where X is the protein in question, a = b = 1, c = 0. For example, at the point of proper removal of the leader peptide by the host cell, the resulting problematic HCV protein can be purified by IMAC (optional). After processing with factor Xa, the protein in question lacks the [(Al) _a- (PS1) _b- (A2) _c ] structure.

  It will be clear that if any of A1, A2, A3, A4, PS1 and PS2 is present, it can be present in a repeating structure. Such a repeating structure still counts as 1 under this circumstance if it exists. That is, a, b, c, d, e, or f is 1, for example, even when A1 exists as, for example, two repetitions (A1-A1).

  By “HCV envelope protein” is meant an HCV E1 or HCV E2 envelope protein or portion thereof that can be derived from an HCV strain of any genotype. More specifically, HCVENV is selected from the group of amino acid sequences consisting of SEQ ID NOs: 85-98 and any fragments thereof, which are amino acid sequences having at least 90% homology with SEQ ID NOs: 85-98. . “Identical” amino acids include multiple groups of conserved amino acids as described above, ie a group consisting of Met, Ile, Leu and Val; a group consisting of Arg, Lys and His; a group consisting of Phe, Trp and Tyr. A group consisting of Asp and Glu; a group consisting of Asn and Gln; a group consisting of Cys, Ser and Thr; and a group consisting of Ala and Gly are considered.

  More specifically, the term “HCV envelope protein” refers to a polypeptide or analog thereof comprising an amino acid sequence (and / or amino acid analog) that defines at least one HCV epitope of either the E1 or E2 region in addition to the glycosylation site. (For example, mimotopes). Both of these envelope proteins can be monomeric, hetero-oligomeric or homo-oligomeric forms of recombinantly expressed envelope protein. Typically, the sequence defining the epitope is in the amino acid sequence of either the E1 or E2 region of HCV (either in the same form or via analog substitution of a native amino acid residue that does not destroy the epitope). Correspond.

  It will be appreciated that the HCV epitope can be co-located with the glycosylation site. In general, the length of the epitope defining sequence will be 3-4 amino acids, more typically 5, 6 or 7, more typically 8 or 9, and even more typically 10 or more. For conformational epitopes, the length of the epitope-defining sequences can vary widely since these epitopes are thought to be formed (eg, folded) by the three-dimensional shape of the antigen. . Thus, the amino acids that define an epitope are relatively small in number, but may be widely distributed along a certain length of the molecule, with folding leading to the proper epitope conformation. . The portion of the antigen between residues that define the epitope may not be as important to the conformational structure of the epitope. For example, deletions or substitutions of these intervening sequences are concomitant provided that sequences critical to epitope conformation are maintained (eg, cysteines involved in disulfide bonds, glycosylation sites, etc.). May not affect the formation epitope. Conformational epitopes can also be formed by two or more essential regions of homo-oligomer or hetero-oligomer subunits.

  As used herein, an epitope of a designated polypeptide refers to an epitope having the same amino acid sequence as an epitope within the designated polypeptide and immunological equivalents thereof. Such equivalents are likewise, for example, strains with currently known sequences, subtype (genotype) or type (group) specific variants or genotypes 1a, 1b, 1c, 1d, 1e, 1f, 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h, 2i, 3a, 3b, 3c, 3d, 3e, 3f, 3g, 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i, 4j, 4k, 4l, 5a, 5b, 6a, 6b, 6c, 7a, 7b, 7c, 8a, 8b, 9a, 9b, 10a, 11 (and its subtype), 12 (and its subtype) or 13 (and its) Strains belonging to (subtype) or any other newly defined HCV (sub) type. The amino acids constituting the epitope need not be part of the linear sequence, and any number of amino acids may be interspersed to form a conformational epitope.

  The HCV antigen of the present invention comprises a conformational epitope derived from the E1 and / or E2 (envelope) domain of HCV. The E1 domain that is thought to correspond to the viral envelope protein is currently estimated to span amino acids 192-383 of the HCV polyprotein, Hijikata et al., 1991). At the time of expression in a (glycosylated) mammalian system, it is believed to have a molecular weight of approximately 35 kDa, as determined via SDS-PAGE. The E2 protein, formerly called NS1, spans amino acids 384-809 or 384-746 of the HCV polyprotein (Grakovi et al., 1993) and is thought to be an envelope protein. At the time of expression in the (glycosylated) vaccinia system, it is believed to have an apparent gel molecular weight of about 72 kDa. It is understood that the end points of these proteins are approximate (eg, the carboxy terminus of E2 is for example amino acids 730, 735, 740, 742, 744, 745, preferably 746, 747, 748, 750, 760, 770 780, 790, 800, 809, 810, 820, and somewhere within the 730-820 amino acid region). The E2 protein is similarly E1, and / or core (aa1-191) and / or P7 (aa747-809) and / or NS2 (aa810-1026) and / or NS3 (aa1027-1657), and / or NS4A (aa1658-1711) and / or NS4B (aa1712-1972) and / or NS5A (aa1973-2420) and / or NS5B (aa2421-1301) and / or any of these HCV proteins different from E2 It can be expressed with these parts. Similarly, E1 protein may be E2 and / or core (aa1-191) and / or P7 (aa747-809) and / or NS2 (aa810-1026) and / or NS3 (aa1027-1657), and / or These HCV proteins that are different from NS4A (aa 1658-1711) and / or NS4B (aa 1712 to 1972) and / or NS5A (aa 1973-2420) and / or NS5B (aa 2421 to 3011) and / or E1 It can also be expressed with any part of Expression in conjunction with these other HCV proteins can be important to obtain proper protein folding.

  As used herein, the term “E1” includes truncated forms and analogs that immunologically cross-react with native E1, and genotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 or any other newly identified HCV type or subtype is included. As used herein, the term “E2” includes truncated forms and analogs that immunologically cross-react with native E2, and genotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 or any other newly identified HCV type or subtype is included. For example, the insertion of multiple codons between codons 383-384 as well as the deletion of amino acids 384-387 has been reported (Kato et al., 1992). Thus, the isolates used in the Examples section of the present invention are not intended to limit the scope of the present invention and HCV 1, 2, 3, 4, 5, 6, 7, 8, It is also understood that any HCV isolate from type 9, 10, 11, 12, or 13 or any other new genotype is a suitable source of E1 and / or E2 sequences for practicing the present invention. Similarly, as described above, the HCV protein that is co-expressed with the HCV envelope protein of the present invention can be derived from any HCV type, and hence from the same type as the HCV envelope protein of the present invention.

  As used herein, “E1 / E2” means an oligomeric form of an envelope protein containing at least one E1 component and at least one E2 component.

The term “specific oligomeric” E1 and / or E2 and / or E1 / E2 envelope protein means all possible oligomeric forms of recombinantly expressed E1 and / or E2 envelope protein that are not aggregates. E1 and / or E2 specific oligomeric envelope proteins are also referred to as homo-oligomer E1 or E2 envelope proteins (see below). The term “single or specific oligomeric” E1 and / or E2 and / or E1 / E2 envelope protein refers to a single monomeric E1 or E2 protein (single in the strict sense) and a specific oligomer E1. And / or E2 and / or E1 / E2 recombinantly expressed protein. These single or specific oligomeric envelope proteins according to the invention can be further defined by the formula (E1) _x (E2) _y . In the formula, x may be a number from 0 to 100 and y may be a number from 0 to 100, provided that both x and y are not 0. When x = 1 and y = 0, the envelope protein includes monomer E1.

  As used herein, the term “homo-oligomer” refers to a complex of E1 or E2 comprising a plurality of E1 or E2 monomers, for example, E1 / E1 dimer, E1 / E1 / E1 three Emer or E1 / E1 / E1 / E1 tetramer and E2 / E2 dimer, E2 / E2 / E2 trimer or E2 / E2 / E2 / E2 tetramer, E1 pentamer and hexamer, All higher order homo-oligomers of E2 pentamer and hexamer or E1 or E2 are all “homo-oligomers” within the scope of this definition. Oligomers include, for example, 1 different E1 or E2 monomers obtained from different types or subtypes of hepatitis C, including those described by both Maertens et al. Two or several can be contained. Such mixed oligomers are still homo-oligomers within the scope of the present invention and may allow a more general diagnosis of HCV.

  The E1 and E2 antigens used in the present invention can be full length viral proteins, substantially full length versions thereof, functional fragments thereof (eg, at least one epitope and / or glycosylation site). Furthermore, the HCV antigens of the present invention can also include other sequences that do not block or prevent the formation of the conformational epitope in question. The presence or absence of a conformational epitope is determined by screening the antigen in question using an antibody (monoclonal or polyclonal sera against the conformational epitope) and reacting with the modified version of the antigen that retains only the linear epitope. And can be easily determined. In such screening using polyclonal antibodies, it may be advantageous to first adsorb polyclonal sera with the denatured antigen and see if it retains antibodies against the antigen in question.

  The HCV protein of the present invention may be glycosylated. A glycosylated protein refers to a protein that contains one or more carbohydrate groups, particularly sugar groups. In general, all eukaryotic cells align six different glycosylation sites on the HCV E1 protein for proper folding and reactivity after aligning different envelope protein sequences of the HCV genotype that can glycosylate the protein. It can be inferred that not all is needed. For example, the HCV subtype 1bE1 protein contains 6 glycosylation sites, but some of these glycosylation sites are not present in certain other (sub) types. The fourth carbohydrate motif (on Asn250) present in types 1b, 6a, 7, 8, and 9 is absent in all other types known today. This glycosylation motif can be mutated to produce a 1bE1-type protein with improved reactivity. Similarly, type 2b sequences show additional glycosylation sites within the V5 region (on Asn299). Isolate S83 belonging to genotype 2C lacks even the first carbohydrate motif in the V1 region even though it is present on all other isolates (Stuyver, L. et al., 1994). However, even between fully conserved glycosylation motifs, the presence of carbohydrates was not required for folding, but may play a role in avoiding immune surveillance mechanisms. Thus, identification of the role of glycosylation can be further tested by mutagenesis of the glycosylation motif. Glycosylation motif mutagenesis (NXS or NXT sequence) is a different amino acid from N when the codon for N, S, or T is N and / or an amino acid different from S or T in the case of S and T Can be achieved by mutating these codons in such a way as to encode. Alternatively, the X position can be mutated to P since NPS or NPT is known not to be frequently modified with carbohydrates. After demonstrating which carbohydrate-addition motifs are required and which are unnecessary for folding and / or reactivity, such mutation combinations can be made. Such experiments have been extensively described by Maertens et al. In WO 96/04385 (Example 8), which is hereby incorporated by reference. As used within the present invention, the term glycosylation means N-glycosylation, unless otherwise specified.

  In particular, the invention relates to core-glycosylated HCV envelope proteins or portions thereof. In this regard, the term “core-glycosylation” refers to a structure “similar” to the structure as depicted by FIG. 3 (box structure) of Hercovices and Orleans (Hercovics, A. and Orleans, P. 1993). Means. Thus, the mentioned carbohydrate structure contains 10 or 11 monosaccharides. In particular, the above disclosure is incorporated herein by reference. The term “similar” means that no more than about 4 additional monosaccharides have been added to the structure, or no more than about 3 monosaccharides have been removed from the structure. . Thus, the carbohydrate structure most preferably consists of 10 monosaccharides but a minimum of 7 monosaccharides, more preferably 8 or 9 monosaccharides, and a maximum of 15, more preferably 14, 13 , 12 or 11 monosaccharides. The included monosaccharide is preferably glucose, mannose or N-acetylglucosamine.

  Another aspect of the present invention covers a vector comprising the polynucleic acid of the present invention or a portion thereof. Such vectors include general purpose cloning vectors such as pUC series or pEMBL series vectors, and are further used in cloning vectors, TA-cloning vectors and Gateway systems (InVitrogen) that require a DNA topoisomerase reaction for cloning. Other cloning vectors such as recombination-based cloning vectors are included. Vectors include plasmids, phagemids, cosmids, bacmids (baculovirus vectors) or can be viral or retroviral vectors. The vector can only function as a cloning means and / or vehicle, otherwise it can further include promoters, enhancers and terminators or polyadenylation signals. The regulatory sequence may allow expression of information contained within the DNA fragment of interest cloned into a vector containing the regulatory sequence. Expression can be the production of RNA or mRNA molecules and the production of the protein molecules. Expression can be the production of RNA molecules using a viral polymerase promoter (eg, SP6, T7 or T3 promoter) introduced at the 5'- or 3'-end of the DNA of interest. Expression can further be transient or stable expression, or alternatively controllable expression. Controllable expression includes inducible expression using, for example, a tetracycline regulatable promoter, a stress-inducible promoter (eg, a human hsp70 gene promoter), a metallothionine promoter, a glucocorticoid promoter, or a progesterone promoter. Bacteria (eg, Escherichia coli, Streptomyces species), insect cells (Spodoptera frugiperda cells, St9 cells), plant cells (eg, potato virus X-based expression vectors, eg, Vance et al., 1998, WO 98/44097) and Expression vectors that mediate expression in mammalian cells (eg, cells from CHO or COS cells, Vero cells, HeLa cell lines) are known.

Thus, this aspect of the invention specifically describes a protein comprising an avian lysozyme leader peptide or a functional equivalent thereof bound to an HCV envelope protein or a portion thereof, or a structure CL-[(A1) _a- (PS1) _b- (A2) _c ] -HCVENV-[(A3) _d- (PS2) _e- (A4) _f ] relates to a vector comprising a recombinant nucleic acid according to the invention which encodes a protein characterized by

  Also implemented in the present invention is the vector further comprising regulatory sequences that allow expression of the protein. In one particular embodiment, the vector following expression is an expression vector. In yet another specific embodiment, the vector according to the invention is an autonomously replicating vector or an integrating vector. In yet another specific embodiment, the vector according to the invention is selected from any of SEQ ID NOs: 20, 21, 32, 35, 36, 39, 40.

  A suitable vector or expression vector of the present invention is a yeast vector. Yeast vectors can contain DNA sequences that allow the vector to replicate autonomously. Examples of such sequences are the yeast plasmid 2μ replication gene REP1-3 and the origin of replication. Other vectors are partially or fully integrated into the enzyme genome. Such integrative vectors are targeted or randomly integrated into specific genomic loci. P. In pastoris, exogenous DN is targeted to the AOXI and HIS4 genes (Gregg, J.M. 1999). In the methanolica, it is targeted to the AUG1 gene (Raymond, CK, 1999). Most recombinant H. coli Within the polymorpha strain, exogenous DNA can be randomly integrated for transformation using a HARS sequence-containing circular plasmid (Hollenberg, CP and Gelissen, G. 1997). Targeted integration includes the MOX / TRP3 locus for analysis / integration (Agaphonov, MO et al. 1995, Sohn, JH et al. 1999), the LEU2 gene (Agaphonov, MO et al. 1999) or rDNA clusters (Cox, H. et al. 2000) can be achieved by homologous recombination. H. Transformation in polymorpha typically results in a variety of individual mitotic stable strains containing a single or multiple copies of the expression cassette in a head-to-tail arrangement. Bring. Strains with up to 100 copies have been identified (Hollenberg, CP and Gelrissen, G. 1997). H. polymorpha or S.P. In the presence of the URA3 gene from C. cerevisiae, the uracil-auxotrophic H. elegans by passage sequence under selective conditions. In polymorpha strain RB11, multiple copies can be randomly integrated. The HARS sequence can be omitted (Gatzke, R. et al., 1995) or can be present (Hollenberg, CP and Gelissen, G. 1997). This passage further leads to a mitotically stable strain. The vector can also include a selectable marker, such as the Schizosaccharomyces pombe TPI gene as described by Russell (Russell, PR 1985), or the yeast URA3 gene. Other marker genes that have been used so far for transformation of Saccharomyces, such as TRP5, LEU2, ADE1, ADE2, HIS3, HIS4, LYS2, can be obtained from, for example, Hansenula, Pichia or Schwannomices.

  A regulatory element (or sequence) that allows expression of the protein in a eukaryotic host is operable with at least one genetic element that represents promoter activity and an open reading frame that encodes the protein to be expressed. It should be understood to include genetic elements that exhibit terminator activity to be bound in any form. The term “promoter” is a nucleotide composed of a consensus sequence that allows binding of RNA polymerase to a DNA template in such a way that mRNA production begins at the normal transcription start site for the adjacent structural gene. Is an array.

  The term “operably linked” means a juxtaposition in a relationship that allows the components thus described to function in their intended manner. A control sequence operably linked to the coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence.

  An “open reading frame” (ORF) is a region of a polynucleotide sequence that encodes a polypeptide and does not include a stop codon. This region may represent a part of the coding sequence or the entire coding sequence.

  A “coding sequence” is a polynucleotide that is translated into a polypeptide and / or transcribed into mRNA when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5 'end and a translation stop codon at the 3' end. A coding sequence can include, but is not limited to, mRNA, DNA (including cDNA) and recombinant polynucleotide sequences.

  A number of regulatory elements are known in the art. Examples of suitable enzyme promoters are Saccharomyces cerevisiae MFa1, TPI, ADHI, ADHII or PGK promoters or corresponding promoters from other enzyme species such as Schizosaccharomyces pombe. Examples of suitable promoters are, for example, (Alber, T. and Kawasaki, G.1982, Ammerer, G.1983, Ballou, L. et al., 1991, Hitzeman, RA, et al., 1980, Kawasaki, G. and Fraenkel, DG 1982, Russell, DW et al., 1983, Russell, PR 1983, Russell, PR and Hall, BD 1983). Suitable yeast terminators are for example the TPI terminators (Albert, T. and Kawasaki, G. 1982) or the yeast CYC1 terminator. For methylotrophic or conditional methylotrophic yeast species, strongly regulatable promoters of enzymes involved in the methanol utilization pathway are excellent candidate promoters, such as the alcohol oxidase gene promoter (Pichia pastoris AOX1, P. methanolica AUG1, Candida It includes AOD1 of bovidini and MOX of Hansenula polymorpha, formaldehyde dehydrogenase promoter (FLD1 of P. pastoris), dihydroxyacetone synthase promoter (DAS1 of C. bodiniii) and formate dehydrogenase (FMD of H. polymorpha). Other promoters are P.I. pastoris or H. p. polymorpha GAP1 promoter and H. coli. polymorpha PMA1 and TPS1 promoters ((Gellissen, G. 2000) and references cited therein). Terminator elements derived from any of these genes are examples of suitable terminator elements, and more particularly, suitable terminator elements include AOD1, AOX1, and MOX terminator elements.

  Another aspect of the present invention covers a host cell comprising a recombinant nucleic acid or vector according to the present invention.

  In a more specific embodiment, a recombinant nucleic acid or vector according to the present invention expresses a protein according to the present invention comprising an avian lysozyme leader peptide or functional variant thereof linked to an HCV envelope protein or a portion thereof. Has ability.

In another embodiment, the host cell has the structure CL-[(A1) _a- (PS1) _b- (A2) _c ] -HCVENV-[(A3) _d- (PS2) _e- (A4) _f ]. Features
In the formula;
CL is an avian lysozyme leader peptide or a functional equivalent thereof;
A1, A2, A3 and A4 are adapter peptides that can be different or the same;
PS1 and PS2 are processing sites that can be different or the same;
HCVENV is an HCV envelope protein or part thereof;
a, b, c, d, e and f are 0 or 1,
Here, A1 and / or A2 may be part of PS1, and / or A3 and / or A4 have the ability to express a protein that may be part of PS2.

  Furthermore, in a particular embodiment of the invention, said host cell comprising a recombinant nucleic acid or vector according to the invention comprises an avian lysozyme leader peptide or a functional equivalent thereof bound to an HCV envelope protein or a part thereof. It has the ability to translocate the containing protein to the endoplasmic reticulum upon removal of the avian lysozyme leader peptide.

In a further specific embodiment thereof, said host cell comprising a recombinant nucleic acid or vector according to the present invention is capable of protein-[(A1) _x- (PS1) _y − into the endoplasmic reticulum upon removal of the CL peptide. (A2) _z ] -HCVENV-[(A3) _x- (PS2) _y- (A4) _z ] has the ability to translocate, and the protein and the CL peptide are CL-[(A1) _a- (PS1 _{) b} - (A2) _{c] -HCVENV - [(A3) d -} (PS2) e - (A4) f] structure characterized by that,
In the formula;
CL is an avian lysozyme leader peptide or a functional equivalent thereof;
A1, A2, A3 and A4 are adapter peptides that can be different or the same;
PS1 and PS2 are processing sites that can be different or the same;
HCVENV is an HCV envelope protein or part thereof;
a, b, c, d, e and f are 0 or 1,
Here, A1 and / or A2 may be a part of PS1, and / or A3 and / or A4 may be a part of PS2.

  Also implemented is a host cell comprising a recombinant nucleic acid or vector according to the invention that has the ability to process the processing sites PS1 and / or PS2 in the protein translocated into the endoplasmic reticulum.

  Also carried out is a host cell comprising a recombinant nucleic acid or vector according to the present invention capable of N-glycosylating said protein translocated to the endoplasmic reticulum.

  Also implemented is a host comprising a recombinant nucleic acid or vector according to the invention that has the ability to N-glycosylate the protein translocated to the endoplasmic reticulum and processed at the sites PS1 and / or PS2. It is a cell.

  More particularly, a host cell comprising a recombinant nucleic acid or vector according to the present invention is a eukaryotic cell, more particularly a yeast cell, for example a Saccharomyces strain such as Saccharomyces cerevisiae cells, Saccharomyces kluyveri or Saccharomyces schavars, Schizosaccharomyces strain, Kluyveromyces lactis, Kluyveromyces strain, Yarrowia lipolytica, Yarrowia strain, Hansenula polymorpha pergillus species, Neurospora strains such as Neurospora crassa, a mutants cells derived from either Schwanniomyces strains such as Schwanniomyces occidentalis cells or thereof.

  The term “eukaryotic cell” includes lower eukaryotic cells as well as higher eukaryotic cells. Lower eukaryotic cells are cells such as yeast cells and fungal cells. Particularly suitable host cells in the context of the present invention are yeast cells or mutant cells derived from any of the above. Mutant cells include yeast glycosylation minus strains, eg, Saccharomyces glycosylation minus strains as used in the present invention. Glycosylation minus strains are defined as strains carrying mutations that result in glycosylation of glycoproteins comparable to core-glycosylation, although the nature of the mutation is not necessarily known. In particular, Saccharomyces glycosylation minus strains are believed to carry mutations that result in significant mobility shifts on the PAGE of the invertase protein. Invertase is a protein that normally exists only in hyperglycosylated form in Saccharomyces (Ballou, L. et al., 1991). Glycosylation minus strains include mnn2, and / or och1 and / or mnn9 deficient strains. The mutant host cell of the present invention is a cell that has lost its ability to remove the avian lysozyme leader peptide from a protein comprising said leader peptide bound to the protein of interest due to the mutation. Not included.

  “Higher eukaryotes” refer to host cells derived from higher animals such as mammals, reptiles, insects and the like. Currently preferred higher eukaryotic host cells are Chinese hamsters (eg C homo-oligomers), monkeys (eg COS and Bellow cells), baby hamster kidney (BHK), pig kidney (PK15), rabbit kidney 13 cells (RK13) human Derived from osteosarcoma cell line 143B, human liver cancer cell lines such as human cell lines HeLa and HepG2, and insect cell lines (eg, Spodoptera frugiperda). Host cells can be provided in suspension or in flask culture, tissue culture, organ culture, and the like. Alternatively, the host cell can be a transgenic animal.

  Introduction of a vector or expression vector into a host cell can be performed by any available transformation or transfection technique applicable to said host cell as is known in the art. Such transformation or transfection techniques include heat shock mediated transformation (eg, E. coli), conjugation DNA transfer, electroporation, PEG mediated DNA uptake, liposome mediated DNA uptake, lipofection, calcium phosphate DNA coprecipitation, DEAE- This includes dextran-mediated transfection, such as direct introduction by microinjection or particle bombardment or introduction with viruses, virions or virus particles.

  Yet another aspect of the present invention is a method for producing an HCV envelope protein or a portion thereof in a host cell, wherein said host cell is transformed with a recombinant nucleic acid according to the present invention or a vector according to the present invention. A method comprising a step, wherein said host cell is capable of expressing a protein comprising an avian lysozyme leader peptide or a functional equivalent thereof bound to an HCV envelope protein or a portion thereof.

In a particular embodiment thereof, a method for producing an HCV envelope protein or part thereof in a host cell comprises the step of transforming said host cell with a recombinant nucleic acid according to the invention or a vector according to the invention. The host cell is characterized by the structure CL-[(A1) _a- (PS1) _b- (A2) _c ] -HCVENV-[(A3) _d- (PS2) _e- (A4) _f ],
In the formula;
CL is an avian lysozyme leader peptide or a functional equivalent thereof;
A1, A2, A3 and A4 are adapter peptides that can be different or the same;
PS1 and PS2 are processing sites that can be different or the same;
HCVENV is an HCV envelope protein or part thereof;
a, b, c, d, e and f are 0 or 1,
Here, A1 and / or A2 may be part of PS1, and / or A3 and / or A4 have the ability to express a protein that may be part of PS2.

In another specific embodiment, the host cell in the method is such that once the CL peptide is removed, the protein CL-[(A1) _a- (PS1) _b ) _b- (A2) _c ]- HCVENV-[(A3) _d- (PS2) _e- (A4) _f ] has the ability to translocate to the endoplasmic reticulum, and the protein and the CL peptide are CL-[(A1) _a- (PS1) _b -(A2) _c ] -HCVENV-[(A3) _d- (PS2) _e- (A4) _f ],
In the formula;
CL is an avian lysozyme leader peptide or a functional equivalent thereof;
A1, A2, A3 and A4 are adapter peptides that can be different or the same;
PS1 and PS2 are processing sites that can be different or the same;
HCVENV is an HCV envelope protein or part thereof;
a, b, c, d, e and f are 0 or 1,
Here, A1 and / or A2 may be part of PS1 and / or A3 and / or A4 is derived from a protein that may be part of PS2.

  Performed similarly is a method for producing an HCV envelope protein or portion thereof wherein the host cell has the ability to N-glycosylate the protein translocated to the endoplasmic reticulum.

  Further implemented is an HCV envelope protein or portion thereof wherein the host cell has the ability to N-glycosylate the protein translocated into the endoplasmic reticulum and processed at the sites PS1 and / or PS2. Is a method for producing

  More particularly, the host cell in any of the above methods for producing an HCV envelope protein or portion thereof is a yeast cell, such as a Saccharomyces strain such as Saccharomyces cerevisiae, Saccharomyces kluyver or Saccharomyces strains, Saccharomyces strains, Kluyveromyces strains such as Kluyveromyces lactis, Yarrowia strains such as Yarrowia lipolytica, Hansengula strains such as Hansenula polymorpha, Pichia pastoris such as Pichia pastoris Strains, Aspergillus species, Neurospora strains such as Neurospora crassa, a mutants cells derived from any such Schwanniomyces strains or of these Schwanniomyces occidentalis.

  Any of the methods according to the invention for producing an HCV envelope protein or part thereof comprises culturing the host cell comprising the recombinant nucleic acid or vector according to the invention in a suitable medium to obtain expression of the protein. Further comprising.

  Further embodiments thereof comprise isolation of the HCV envelope protein produced from the host cell or alternatively from the host cell or a portion thereof. The isolation step comprises: (i) degradation of the host cell in the presence of a chaotropic agent; (ii) chemical and / or enzyme of a cysteine thiol group in the isolated protein. One or more of the following modifications may be included: (modification may be reversible or irreversible, producing HCV envelope protein or portion thereof) and (iii) heparin affinity chromatography.

  Examples of “chaotropic agents” are guanidinium chloride and urea. In general, chaotropic agents are chemical substances that can break the hydrogen bond structure of water. In concentrated solutions, they can denature proteins from reducing the hydrophobic effect.

In an HCV envelope protein or portion thereof as described herein comprising at least one cysteine residue, but preferably two or more cysteine residues, the thiol group of cysteine is a chemical or enzymatic means Can be irreversibly protected. In particular, “irreversible protection” or “irreversible blocking” by chemical means involves alkylation, preferably an HCV envelope using an alkylating agent such as active halogen, ethyleneimine or N- (iodoethyl) trifluoro-acetamide. It means alkylation of protein. In this regard, alkylation of the cysteine thiol group is (CH ₂ ) _n R, where n is 0, 1, 2, 3 or 4 and R is H, COOH, NH ₂ , CONH ₂ , phenyl. It should be understood to mean the replacement of the thiol hydrogen with (or any derivative thereof). Alkylation can be performed by any method known in the art such as, for example, active halogen X (CH ₂ ) _n R, where X is a halogen such as I, Br, Cl or F. Examples of active halogens are methyl iodide, iodoacetic acid, iodoacetamide, and 2-bromoethylamine. Other methods of alkylation, NEM (N- ethylmaleimide) or biotin -NEM, mixtures or, ethyleneimine results in the substitution of -H by both _{-CH 2 -CH} 2 -NH 2 as a result or N- The use of (iodoethyl) trifluoroacetamide is included (Hermanson, GT 1996). The term “alkylating agent” as used herein means a compound capable of performing an alkylation as described herein. Such alkylation ultimately results in a modified cysteine that can mimic other amino acids. Alkylation with ethyleneimine results in a structure that resembles lysine, thus introducing a new cleavage site for trypsin (Hermanson, GT 1996). Similarly, utilization of methyl iodide results in amino acids resembling methionone, while utilization of iodoacetate and iodoacetamide results in amino acids resembling glutamic acid and glutamine, respectively. Similarly, these amino acids are preferably used in direct mutation of cysteine. Accordingly, the present invention is described herein wherein at least one cysteine residue of an HCV envelope protein as described herein is mutated to a natural amino acid, preferably methionine, glutamic acid, glutamine or lysine. Related to the HCV envelope protein. The term “mutated” is described in site-directed mutagenesis of nucleic acids encoding these amino acids, ie, site-directed mutagenesis using, for example, PCR or (Sambrook, J. et al., 1989). Means methods well known in the art, such as via oligonucleotide-mediated mutagenesis. For the Examples section of the present invention, unless otherwise specified, alkylation should be understood to mean the use of iodo-acetamide as an alkylating agent.

Furthermore, it is understood that the cysteine thiol group of the HCV protein of the present invention or a portion thereof can be reversibly protected. The purpose of reversible protection is to stabilize the HCV protein or part thereof. In particular, after reversible protection, sulfur-containing functional groups (eg thiols and disulfides) are retained under non-reactive conditions. Sulfur-containing functional groups are thus unable to react with other compounds and have, for example, no tendency to form or exchange disulfide bonds as follows.

  The described reactions between thiol and / or disulfide residues are not limited to intermolecular processes, but can also occur intramolecularly.

  As used herein, the terms “reversibly protect” or “reversibly block” refer to covalently attaching a modifying agent to a thiol of cysteine and purifying the redox state of the thiol group. It is intended to manipulate (shield) the HCV protein environment in such a way that it remains unaffected throughout the subsequent steps of the procedure.

  Reversible protection of the cysteine thiol group can be performed chemically or enzymatically.

  As used herein, “reversible protection by enzymatic means” is mediated by an enzyme such as an acyl-transferase, such as an acyltransferase involved in acting as a catalyst for thio-esterification, such as, for example, palmitoyl acyltransferase. Intended for reversible protection (see below).

（Ｄａｒｂｒｅ，Ａ．１９８６）又は

（亜硫酸分解： Ｋｕｍａｒ，Ｎ．ら、１９８６）。スルホン化のための試薬は、例えばＮａ_２ＳＯ_３又はテトラチオン酸ナトリウムである。後者のスルホン化試薬は、１０〜２００ｍＭ、より好ましくは５０〜２００ｍＭの濃度で使用される。スルホン化は、例えばＣｕ^２＋（１００μＭ〜１ｍＭ）又はシステイン（１〜１０ｍＭ）のごとき触媒の存在下で実施可能である。
反応は、タンパク質変性ならびに未変性条件下で実施可能である（Ｋｕｍａｒ，Ｎ．ら、１９８５；Ｋｕｍａｒ，Ｎ．ら、１９８６）
チオエステル結合形成又はチオエステル化は、以下の式によって特徴づけされる：

なお式中、Ｘは好ましくは化合物Ｒ’ＣＯ−Ｘ内のハロゲニドである。
２．例えば重金属、特にＺｎ^２＋、Ｃｄ^２＋、モノ−、シチオ及びジスルフィド−化合物（例えばアリル−及びアルキルメタンチオスルホネート、ジチオピリジン、ジチオモルフォリン、ジヒドロリポアミン、エルマン試薬、アルドロチオール^ＴＭ（アルドリッチ（Ａｌｄｒｉｃｈ））（Ｒｅｉｎ，Ａ．ら、１９９６）、ジチオカルバメート）、又はチオール化剤（例えばグルタチオン、Ｎ−アセチルシステイン、システインアミン）などにより本発明のシステイニルを可逆的に修飾する修飾用作用物質による。ジチオカルバメートは、スルフヒドリル基と反応する能力を付与するＲ_１、Ｒ_２ＮＣ（Ｓ）ＳＲ_３官能基を有する広範なクラスの分子を含んで成る。チオール含有化合物が、好ましくは０．１〜５０ｍＭ、より好ましくは１〜５０ｍＭ、さらに一層好ましくは１０〜５０ｍＭの濃度で使用される。
３．１０μＭ〜１０ｍＭ、より好ましくは１〜１０ｍＭの濃度範囲内での例えばＤＴＴ、ジヒドロアスコルベート、ビタミンＳ及び誘導体、マンニトール、アミノ酸、ペプチド及び誘導体（例えばヒスチジン、エルゴチオネイン、カルノシン、メチオニン）、没食子酸塩、ヒドロキシアニソール、ヒドロキシトルエン、ハイドロキノン、ヒドロキシメチルフェノール及びその誘導体のごとき、チオール状態を保存する（安定化させる）修飾用作用物質、特に酸化防止剤の存在による。
４．例えば（ｉ）金属イオン（Ｚｎ^２＋、Ｍｇ^２＋）、ＡＴＰのごとき補因子、（ｉｉ）ｐＨ制御（例えば、タンパク質については大部分のケースにおいてｐＨ〜５又はｐＨは好ましくはチオールｐＫ_ａ−２；例えば逆相クロマトグラフィーにより精製されたペプチドについてはｐＨ〜２に）のごときチオール安定化条件による。 As used herein, “reversible protection by chemical means” intends reversible protection by:
1. With modifiers that reversibly modify cysteinyl, for example by sulfonation and thioesterification:
Sulfonation is a reaction in which thiols or cysteines involved in disulfide bridges are modified to S-sulfonates:

(Darbre, A. 1986) or

(Sulphite degradation: Kumar, N. et al., 1986). Reagents for sulfonation are for example Na ₂ SO ₃ or sodium tetrathionate. The latter sulfonation reagent is used at a concentration of 10 to 200 mM, more preferably 50 to 200 mM. Sulfonation can be carried out in the presence of a catalyst such as Cu ²⁺ (100 μM to 1 mM) or cysteine (1 to 10 mM).
The reaction can be performed under protein denaturing as well as native conditions (Kumar, N. et al., 1985; Kumar, N. et al., 1986).
Thioester bond formation or thioesterification is characterized by the following formula:

In the formula, X is preferably a halogenide in the compound R′CO—X.
2. For example heavy metals, especially ^Zn 2 +, ^{Cd 2+,} mono -, Shichio and disulfide - compounds (such as allyl - and alkyl thiosulfonate, dithiopyridine, dithio morpholine, dihydrolipoamide amine, Ellman's reagent, Al mud thiol ^TM (Aldrich ( Aldrich)) (Rein, A. et al., 1996), dithiocarbamate), or by a modifying agent that reversibly modifies the cysteinyl of the present invention with a thiolating agent (eg, glutathione, N-acetylcysteine, cysteine amine), etc. . Dithiocarbamates comprise a broad class of molecules with R ₁ , R ₂ NC (S) SR ₃ functional groups that confer the ability to react with sulfhydryl groups. The thiol-containing compound is preferably used at a concentration of 0.1-50 mM, more preferably 1-50 mM, even more preferably 10-50 mM.
3. DTT, dihydroascorbate, vitamin S and derivatives, mannitol, amino acids, peptides and derivatives (eg histidine, ergothioneine, carnosine, methionine), gallic acid within a concentration range of 10 μM to 10 mM, more preferably 1 to 10 mM By the presence of modifying agents, particularly antioxidants, that preserve (stabilize) the thiol state, such as salts, hydroxyanisole, hydroxytoluene, hydroquinone, hydroxymethylphenol and derivatives thereof.
4). For example (i) metal ions ^{(Zn 2+,} Mg ^2+), such as cofactor for ATP, (ii) pH control (e.g., pH ~ 5 or pH in most cases for the protein is preferably a thiol _pK a -2; For example, for peptides purified by reverse phase chromatography, to thiol stabilization conditions such as pH˜2.

  The combination of reversible protection as described in (1), (2), (3), and (4) may result in HCV proteins that are equally pure and regenerated. In practice, a combination compound, such as Z103 (Zn carnosine), can be used, preferably at a concentration of 1-10 mM. Reversible protection also means any cysteinyl protection method that can be reversed enzymatically or chemically without disrupting the peptide backbone, in addition to the modifying groups or shielding described above. In this regard, the present invention specifically describes, for example, that the thioester bond is thioesterase, basic buffer conditions (Beekman, NJ et al., 1997) or hydroxylamine treatment (Vingerhoeds, MH et al., 1996). Specifically based on peptides prepared by conventional chemical synthesis (see above), cleaved by.

  Thiol-containing HCV proteins include, for example, (1) cleavable connector arms containing disulfide bonds (eg, immobilized 5,5′-dithiobis (2-nitrobenzoic acid) (Jayabaskaran, C. et al., 1987) and activity Can be purified on affinity chromatography resin containing electron hexavalent thiol-sepharose 4B (Pharmacia) or (2) aminohexanoyl-4-aminophenylarsine as immobilized ligand. is there. The latter affinity matrix has been used for proteins subject to redox regulation and dithiol proteins that are targets for oxidative stress (Kalev, E. et al., 1993).

  Reversible protection can also be used to increase peptide solubilization and extraction (Pomroy, NC & Deber, CM 1998).

  The reversible protection and thiol stabilizing compound can be presented in the form of a monomer, polymer or liposome.

Removal of the reversible protection state of cysteine residues can be achieved chemically or enzymatically, for example by:
- particularly 1 to 200 mM, more preferably the reducing agent at a concentration of 50 to 200 mM, in particular DTT, DTE,-2-mercaptoethanol, dithionite, SnCl _2, sodium hydride borate, hydroxylamine, TCEP;
-Removal of thiol stabilization conditions or agents eg by increasing pH;
An enzyme such as in particular thioesterase, glutaredoxin, thioredoxin, in particular in the concentration range of 0.01-5 μM, more particularly 0.1-5 μM;
A combination of the chemical and / or enzymatic conditions described above.

  Removal of the reversible protection state of cysteine residues can be performed in vivo or in vitro, for example, in a cell or in an individual.

  Within the purification procedure, cysteine residues may or may not be irreversibly blocked and may or may not be replaced by any reversible modifying agent as listed above. You will understand.

The reducing agent according to the invention is any agent that achieves the reduction of sulfur in cysteine residues, such as “SS” disulfide bridges, desulfonation of cysteine residues (RS—SO ₃ ⁻ → RSH). Antioxidants are any reagent that preserves the thiol state or preserves “SS” formation and / or exchange. Reduction of the “S—S” disulfide bridge is a chemical reaction that reduces the disulfide to a thiol (—SH). The disulfide bridge breaking agents and methods disclosed by Maertens et al. In WO 96/04385 are hereby incorporated herein by reference. “S—S” reduction can be obtained (1) by an enzymatic cascade or (2) by a reducing compound. Enzymes such as thioredoxin and glutaredoxin are known to be involved in the in vivo reduction of disulfides and have also been shown to be effective in reducing "SS" bridges in vitro. Disulfide bond, the secondary rate of ¹⁰⁴ times back and forth greater apparent than the corresponding rate constant for the reaction with DTT, are rapidly cleaved by reduced already thioredoxin at pH 7.0. The reduction kinetics can be dramatically increased by preincubating the protein solution with 1 mM DTT or dihydrolipoamide (Holmgren, A. 1979). Thiol compounds capable of reducing protein disulfide bridges include, for example, dithiothreitol (DTT), dithioerythritol (DTE), β-mercaptoethanol, thiocarbamate, bis (2-mercaptoethyl) sulfone and N, N′-bis. (Mercaptoacetyl) hydrazine and sodium dithionite. For the reduction of HCV proteins, it has been shown to be very useful in the reduction of disulfide bridges within monoclonal antibodies (Thakur, ML et al., 1991), ascorbate or stannous chloride (SnCl A reducing agent having no thiol group as in ₂ ) can also be used. Furthermore, changes in pH value can affect the redox state of HCV proteins. Sodium borohydride treatment has been shown to be effective for the reduction of disulfide bridges within peptides (Gailit, J. 1993). Tris (2-carboxyethyl) phosphine (TCEP) can reduce disulfides at low pH (Burns, J. et al., 1991). When DTT or sodium borohydride is used as the reducing agent, selenol acts as a catalyst for disulfide to thiol reduction. A commercially available diselenide, selenocysteamine, was used as the catalyst precursor (Singh, R. and Kats, L. 1995).

  Heparin is known to bind to multiple viruses and therefore binding to the HCV envelope has already been suggested (Garson, JA et al., 1991). In this regard, to analyze the potential binding of HCV envelope protein to heparin, heparin can be biotinylated and then the interaction of HCV envelope protein with heparin is observed, for example, on a microtiter plate coated with HCV envelope protein. Can be analyzed. In this way, different expression systems can be examined closely. For example, strong binding to a portion of HCV E1 expressed in Hansenula has been observed while there is no binding at HCV E1 from mammalian cell culture. In this regard, the term “heparin affinity chromatography” relates to immobilized heparin that can specifically bind to HCV envelope proteins. High mannose type proteins bind agglutinins such as Lens culinaris, Galanthus nivariis, Narcissus pseudonarcissus, Pisum sativum or Allium ursinum. Furthermore, N-acetylglucosamine can be bound by lectins such as WGA (wheat germ agglutinin) and equivalents thereof. Thus, for the purpose of separating the HCV envelope protein of the present invention from cell culture supernatants, cell lysates and other fluids, for example for the purpose of purification during the production of antigens for vaccines or immunoassays, a lectin bound to a solid phase is used. Can be used.

  “HCV recombinant vaccinia virus” means a vaccinia virus comprising a nucleic acid sequence encoding an HCV protein or a portion thereof.

A further aspect of the invention relates to an isolated HCV envelope protein or portion thereof resulting from a production method as described herein. In particular, the present invention provides a recombinant nucleic acid comprising a nucleotide sequence encoding a protein comprising an avian lysozyme leader peptide or a functional equivalent thereof bound to an HCV envelope protein or a portion thereof, in a eukaryotic cell. Relates to the isolated HCV envelope protein or part thereof resulting from expression in More particularly, the recombinant nucleic acid, CL - _{[(A1) a - (PS1)} b - (A2) c ] _{-HCVENV - [(A3) d -} (PS2) e - (A4) f 4) _f ]],
In the formula;
CL is an avian lysozyme leader peptide or a functional equivalent thereof;
A1, A2, A3 and A4 are adapter peptides that can be different or the same;
PS1 and PS2 are processing sites that can be different or the same;
HCVENV is an HCV envelope protein or part thereof;
a, b, c, d, e and f are 0 or 1,
Here, A1 and / or A2 encodes a protein that may be part of PS1 and / or A3 and / or A4 may be part of PS2.

In one particular embodiment, the isolated HCV envelope protein or portion thereof is derived from said protein comprising an avian lysozyme leader peptide or a functional equivalent thereof bound to said HCV envelope protein or portion thereof. Is done. In another specific embodiment, the isolated HCV envelope protein or a portion thereof is CL-[(A1) _a- (PS1) _b- (A2) _c ] -HCVENV-[(A3) _d- ( PS2) _e − (A4) _f ]
In the formula;
CL is an avian lysozyme leader peptide or a functional equivalent thereof;
A1, A2, A3 and A4 are adapter peptides that can be different or the same;
PS1 and PS2 are processing sites that can be different or the same;
HCVENV is an HCV envelope protein or part thereof;
a, b, c, d, e and f are 0 or 1,
Wherein A1 and / or A2 may be part of PS1 and / or A3 and / or A4 is derived from said protein which may be part of PS2.

  Another aspect of the invention relates to the use of an avian lysozyme leader peptide to direct a recombinantly expressed protein to the endoplasmic reticulum of Hansenula polymorpha or any mutant thereof.

  Thus, all aspects and embodiments of the invention as described above and with respect to the HCV envelope protein, include H. as host cells. Specifically for polymorpha or any mutant thereof, it can be read as for one protein rather than for the HCV envelope protein.

  More particularly, the present invention also relates to a recombinant nucleic acid comprising a nucleotide sequence encoding a protein comprising an avian lysozyme leader peptide or a functional equivalent thereof linked to the protein of interest or a portion thereof. .

In one embodiment thereof, the recombinant nucleic acid comprising a nucleotide sequence is
CL-[(A1) _a- (PS1) _b- (A2) _c ] -PROT-[(A3) _d- (PS2) _e- (A4) _f ]
It is characterized by the structure
In the formula;
CL is an avian lysozyme leader peptide or a functional equivalent thereof;
A1, A2, A3 and A4 are adapter peptides that can be different or the same;
PS1 and PS2 are processing sites that can be different or the same;
PROT is the protein of interest or part thereof;
a, b, c, d, e and f are 0 or 1,
Here, A1 and / or A2 encode a protein which may be part of PS1 and / or A3 and / or A4 may be part of PS2.

In a further embodiment, the recombinant nucleic acid according to the present invention further comprises a protein comprising the avian lysozyme leader peptide or a functional equivalent thereof linked to the protein of interest or part thereof, or the structure CL-[( A1) _x- (PS1) _y- (A2) _z ] -PROT-[(A3) _x- (PS2) _y- (A4) _z ]] It comprises regulatory elements that allow expression in polymorpha cells or mutants thereof. Further included are a vector comprising the recombinant nucleic acid, a host cell comprising the recombinant nucleic acid or the vector, an avian lysozyme leader peptide or functional variant thereof linked to the protein of interest. Said host cell expressing the protein comprising.

A further aspect of the present invention provides a recombinant nucleic acid in a Hansenula cell comprising a nucleotide sequence encoding a protein comprising an avian lysozyme leader peptide or a functional equivalent thereof linked to the protein of interest or a portion thereof. Relates to an isolated protein of interest or part thereof obtained as a result of expression in More specifically, the recombinant nucleic acid has a structure of CL-[(A1) _a- (PS1) _b- (A2) _c ] -PROT-[(A3) _d- (PS2) _e- (A4) _f ]. Features
In the formula;
CL is an avian lysozyme leader peptide or a functional equivalent thereof;
A1, A2, A3 and A4 are adapter peptides that can be different or the same;
PS1 and PS2 are processing sites that can be different or the same;
PROT is the protein of interest or part thereof;
a, b, c, d, e and f are 0 or 1,
Here, A1 and / or A2 encodes a protein that may be part of PS1 and / or A3 and / or A4 may be part of PS2.

In one particular embodiment, the isolated protein of interest or portion thereof is derived from said protein comprising an avian lysozyme leader peptide or functional equivalent thereof linked to the protein of interest or portion thereof. . In another specific embodiment, the isolated protein of interest or portion thereof is CL-[(A1) _a- (PS1) _b- (A2) _c ] -PROT-[(A3) _d- ( PS2) _e − (A4) _f ]
In the formula;
CL is an avian lysozyme leader peptide or a functional equivalent thereof;
A1, A2, A3 and A4 are adapter peptides that can be different or the same;
PS1 and PS2 are processing sites that can be different or the same;
PROT is the protein of interest or part thereof;
a, b, c, d, e and f are 0 or 1,
Here A1 and / or A2 may be part of PS1 and / or A3 and / or A4 is derived from said protein which may be part of PS2.

  In particular embodiments of the invention, the protein of interest or fragment thereof may be a viral envelope protein or fragment thereof, such as, for example, HCV envelope protein or HBV (hepatitis B) envelope protein or fragment thereof. In general, the protein of interest or fragment thereof can be any protein that requires the N-glycosylation properties of the present invention. Examples of other virus envelope proteins include HIV (human immunodeficiency virus) envelope protein gp120 and virus envelope proteins of viruses belonging to Flavivideae.

  The terms “HCV virus-like particles formed from HCV envelope proteins” and “oligomer particles formed from HCV envelope proteins” are here composed of one or two E1 and / or E2 monomers, respectively. Is defined as a structure of particular nature and shape that contains multiple basic units of HCV E1 and / or E2 envelope proteins. It should be clear that the particles of the invention are defined as lacking an infectious HCV RNA genome. It may be a spherical, higher order particle, which may be empty, consisting of a shell of an envelope protein into which the particles, lipids, surfactants, HCV core protein or adjuvant molecules of the invention can be incorporated. The latter particles can likewise be encapsulated by liposomes or apolipoproteins such as apolipoprotein B or low density lipoproteins or by any other means of targeting the particles to specific organs or tissues. In this case, such empty spherical particles are often referred to as “virus-like particles” or VLPs. Alternatively, the higher order particles may have complete spheres composed of HCV E1 or E2 envelope protein oligomers, which may further incorporate lipids, surfactants, HCV core proteins or adjuvant molecules Or a solid globular structure that can itself be embedded by liposomes such as apolipoprotein B, low density lipoproteins or any other means of targeting the particles to specific organs or tissues such as apolipoproteins or asialoglycoproteins, for example. possible. The particles may also be composed of smaller structures (compared to the empty or solid spherical structures described above) that are usually round (see below) and usually contain only a single layer of HCV envelope protein. is there. A standard example of such a small particle is a rosette-like structure consisting of a lower number of HCV envelope proteins, usually between 4-16. A specific example of the latter includes smaller particles obtained with E1 in 0.2% CHAPS as exemplified herein, apparently containing 8-10 E1 monomers. Such rosette-like structures are usually organized in one plane and have a round shape, for example a wheel shape. Again, lipids, surfactants, HCV core proteins, or adjuvant molecules can be further incorporated, or by liposomes or apolipoproteins such as apolipoprotein B or low density lipoproteins or in specific organs. Alternatively, smaller particles can be embedded by any other means of targeting the particles to tissue. Smaller particles can likewise be further incorporated with lipids, surfactants, HCV core proteins, or adjuvant molecules, and by themselves liposomes or apolipoproteins such as apolipoprotein B or low density lipoprotein. Or can form small spherical or small spherical structures of similar fewer HCV E1 or E2 envelope proteins that can be embedded by any other means of targeting the particles to specific organs or tissues . The size (ie, diameter) of the above-mentioned particles as measured by dynamic light scattering techniques well known in the art (see further examples section) is usually between 1 and 100 nm, more preferably between 2 and It is between 70 nm, even more preferably between 2-40 nm, 3-20 nm, 5-16 nm, 7-14 nm or 8-12 nm.

In particular, the present invention is a method for purifying hepatitis C virus (HCV) envelope protein or any portion thereof suitable for use in immunoassays or vaccines:
(I) growing Hansenula or Saccharomyces glycosylation minus strains transformed with an envelope gene encoding HCV E1 and / or HCV E2 protein or any portion thereof in an appropriate medium;
(Ii) causing expression of the HCV E1 and / or HCV E2 gene or any portion thereof; and (iii) purifying the HCV E1 and / or HCV E2 protein or portion thereof from the cell culture;
A method comprising:

The present invention is further a method for purifying hepatitis C virus (HCV) envelope protein or any portion thereof suitable for use in immunoassays or vaccines:
(I) growing Hansenula or Saccharomyces glycosylation minus strains transformed with an envelope gene encoding HCV E1 and / or HCV E2 protein or any portion thereof in an appropriate medium;
(Ii) causing expression of the HCV E1 and / or HCV E2 gene or any part thereof; and (iii) transforming the HCV E1 and / or HCV E2 protein or part thereof expressed in the cell. Purification after lysis of the host cells
Related to a method comprising:

The present invention is further a method for purifying hepatitis C virus (HCV) envelope protein or any portion thereof suitable for use in immunoassays or vaccines:
(I) growing Hansenula or Saccharomyces glycosylation minus strains transformed with an envelope gene encoding HCV E1 and / or HCV E2 protein or any portion thereof comprising at least two Cys-amino acids in a suitable medium;
(Ii) causing expression of the HCV E1 and / or HCV E2 gene or any portion thereof, and (iii) the Cys amino acid is reversibly protected by chemical and / or enzymatic means, Purifying HCV E1 and / or HCV E2 protein or a portion thereof from said cell culture;
Related to a method comprising:

The present invention is further a method for purifying hepatitis C virus (HCV) envelope protein or any portion thereof suitable for use in immunoassays or vaccines:
(I) growing Hansenula or Saccharomyces glycosylation minus strains transformed with an envelope gene encoding HCV E1 and / or HCV E2 protein or any portion thereof comprising at least two Cys-amino acids in a suitable medium;
(Ii) causing expression of the HCV E1 and / or HCV E2 gene or any part thereof, and (iii) the Cys amino acid is reversibly protected by chemical and / or enzymatic means, Purifying the intracellularly expressed HCV E1 and / or HCV E2 protein or a portion thereof after lysis of the transformed host cell has occurred;
Related to a method comprising:

  The present invention relates to a method for purifying recombinant HCV yeast protein, or any portion thereof, as described herein, wherein purification includes heparin affinity chromatography.

  Accordingly, the present invention also relates to a method for purifying a recombinant yeast protein or any part thereof as described above, wherein said chemical means is sulfonation.

  Accordingly, the present invention also relates to a recombinant HCV yeast protein, as described above, wherein said reversible protection of Cys-amino acids is replaced with irreversible protection by chemical and / or enzymatic means. It also relates to a method for purifying any part thereof.

  Accordingly, the present invention also relates to a method for purifying recombinant HCV yeast protein or any part thereof, as described above, wherein said irreversible protection by chemical means is iodoacetamide. .

  Accordingly, the present invention also purifies recombinant HCV yeast protein or any portion thereof, as described above, wherein the irreversible protection by chemical means is NEM or biotin-NEM or mixtures thereof. Also relates to a method for

  The invention also relates to a composition as described above which also comprises an HCV core, E1, E2, P7, NS2, NS3, NS4A, NS4B, NS5A and / or NS5B protein or part thereof. The core-glycosylated proteins E1, E2, and / or E1 / E2 of the present invention can also be combined with other HCV antigens such as core, P7, NS3, NS4A, NS4B, NS5A and / or NS5B, for example. Purification of these NS3 proteins will preferably involve reversible modification of cysteine residues, and even more preferably cysteine sulfonation. Methods for obtaining such reversible modifications, including sulfonation, have been described for the NS3 protein in Mertens et al. PCT / EP99 / 02547. It should be emphasized that the entire contents, including all definitions in the latter document, are incorporated herein by reference.

  Similarly, the present invention relates to an envelope protein as described herein for inducing immunity against HCV, characterized in that the envelope protein is used as part of a series of times and compounds. About the use of. In this regard, the term “a series of times and compounds” should be understood to mean that the compound used to elicit an immune response is administered to an individual at intervals of time. The latter compound can comprise any of the following components: HCV envelope protein, HCV DNA vaccine composition, HCV polypeptide according to the present invention.

In this regard, one series includes administration as follows:
(I) administration of the HCV envelope protein according to the present invention at time intervals, or (ii) different times, including the envelope protein and the HCV DNA vaccine composition at the same time or alternating time intervals. Administration of HCV antigens, such as, for example, HCV envelope proteins according to the invention, in combination with HCV DNA vaccine compositions that can be administered at regular intervals, or (iii) other HCVs at intervals of time Either (i) or (ii) in combination with a peptide.

  In this regard, the HCV DNA vaccine composition comprises a nucleic acid encoding an E1-, E2-, E1 / E2-peptide, NS3 peptide, other HCV peptide or HCV envelope peptide including a portion of said peptide. Should be obvious. In addition, it should be understood that the HCV peptides comprise HCV envelope peptides including E1-, E2-, E1 / E2-peptides, other HCV peptides or portions thereof. The term “other HCV peptide” means any HCV peptide or fragment thereof. In item II of the above scheme, the HCV DNA vaccine composition preferably comprises a nucleic acid encoding an HCV envelope peptide. In item II of the above scheme, the HCV DNA vaccine composition is even more preferably composed of a nucleic acid encoding an HCV envelope peptide, optionally in combination with an HCV-NS3 DNA vaccine composition. In this regard, it should be apparent that the HCV DNA vaccine composition comprises a plasmid vector comprising a polynucleotide sequence encoding an HCV peptide as described above operably linked to a transcriptional regulatory element. . As used herein, “plasmid vector” means a nucleic acid molecule that has the ability to transport another nucleic acid to which it has been linked. In general, although not limiting, plasmid vectors are circular double stranded DNA loops that are not linked to a chromosome in their vector form. As used herein, “polynucleotide sequence” refers to a polynucleotide, such as deoxyribonucleic acid (DNA) and, where applicable, ribonucleic acid (RNA). This term is to be understood as including nucleotide analogs and analogs of either RNA or DNA made from single-stranded (sense or antisense) and double-stranded polynucleotides. As used herein, the term “transcriptional regulatory element”, when introduced into a living vertebrate cell, directs a cellular mechanism to produce a translation product encoded by the polynucleotide. It means a nucleotide sequence containing essential regulatory elements in such a way that it can. The term operatively linked means a juxtaposition in which the constituent elements are configured to perform their normal functions. Thus, a transcriptional regulatory element operably linked to a nucleotide sequence has the ability to effect expression of the nucleotide sequence. One skilled in the art will recognize that different transcription promoters, terminators, carrier vectors or specific gene sequences can be successfully used.

  Alternatively, the DNA vaccine can be delivered through a live vector such as adenovirus, canary variola virus, MVA and the like.

  The HCV envelope protein of the present invention or a part thereof is particularly suitable for incorporation into immunoassays for detection of anti-HCV antibodies and / or genotyping of HCV, for diagnosis / monitoring of HCV diseases or as therapeutic agents. .

  A further aspect of the invention is a diagnostic kit for detecting the presence of an anti-HCV antibody in a sample suspected of containing an anti-HCV antibody, comprising an HCV envelope protein according to the invention or a part thereof. Relates to a kit consisting of In one particular embodiment thereof, the HCV envelope protein or portion thereof is attached to a solid support. In a further embodiment, the sample suspected of containing anti-HCV antibodies is a biological sample.

  As used herein, the term “biological sample” refers to serum, plasma, lymph, skin, airways, intestinal tract and external sections of the genitourinary tract, oocytes, tears, saliva, milk, blood cells, tumors, organs, stomach A sample of tissue or fluid isolated from an individual, including (but not limited to) exocrine secretions such as secretions, mucus, spinal fluid such as excreta, urine, semen and the like.

  Another aspect of the invention relates to a composition comprising an isolated HCV envelope protein or fragment thereof according to the invention. The composition can further comprise a pharmaceutically acceptable carrier and can be a drug or a vaccine.

  A further aspect of the invention covers a medicament or vaccine comprising an HCV envelope protein according to the invention or a part thereof.

  Yet another aspect of the present invention includes an effective amount of an HCV envelope protein according to the present invention or a portion thereof and a pharmaceutically acceptable adjuvant in a pharmaceutical composition for inducing an HCV specific immune response in a mammal. A pharmaceutical composition comprising: Said pharmaceutical composition comprising an effective amount of an HCV envelope protein or part thereof according to the invention likewise has the ability to induce HCV-specific antibodies in the mammalian body or to induce T cell function in the mammalian body. You can also. Said pharmaceutical composition comprising an effective amount of an HCV envelope protein according to the invention or a part thereof may be a prophylactic composition or a therapeutic composition. In certain embodiments, the mammal is a human.

  "Mammal" should be understood as any member of the higher vertebrate mammal network, including humans, characterized by the fact that females have mammary glands that secrete live birth, body hair, and milk for raising children . Thus, mammals also include non-human primates and trimera mice (Zaukerman et al., 1999).

  “Vaccine” or “drug” refers to the ability to elicit protection against a disease, whether partial or complete, and whether it is against acute or chronic disease A composition having In this case, the vaccine or drug is a prophylactic vaccine or drug. A vaccine or drug may also be useful for treating an already ill individual, in which case it is referred to as a therapeutic vaccine. Similarly, the pharmaceutical composition can be used for any prophylactic and / or therapeutic application, in which case it is a prophylactic and / or therapeutic composition, respectively.

  The HCV envelope protein of the present invention is intact, in biotinylated form (as described in WO 93/18054) and / or Neutralite Avidin '(Molecular Probes Inc., Eugene, Oreg., USA), avidin Alternatively, it can be used in a complexed state with streptavidin. Also suitable vaccines, diluents, in which the “vaccine” or “drug”, in addition to the active substance, alone does not induce the production of antibodies harmful to the individual receiving the composition and does not cause protection, It should also be pointed out that it comprises a “pharmaceutically acceptable carrier” or “pharmaceutically acceptable adjuvant” which can be a carrier and / or an adjuvant. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers and inactive virus particles. Such carriers are well known to those skilled in the art. Preferred adjuvants for enhancing the effectiveness of the composition include, without limitation, the following: Aluminum hydroxide, 3-O-deacylation as described in WO 93/19780 Monoformyl lipid A, aluminum phosphate as described in WO 93/24143, N-acetyl-muramyl-L-threonine-D-isoglutamine as described in US Pat. No. 4,606,918, N -Acetyl-normuramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine 2- (1'2 'dipalmitoyl-sn-glycero-3-hydroxyphosphoryl Oxy) ethylamine, monophosphoryl lipid A, detoxified endotoxin, trehalose-6 - Jimikore - DOO and cell wall skeleton (MPL + TDM + CWS) 2% squalene / Tween 80 RIBI contained in the emulsion (Immuno Chem Research Inc., Hamilton Montana, USA). Any of the three components MPL, TDM or CWS can be used alone or in combination. MPL, RIBI. It can also be replaced by its synthetic analog called 529. In addition, Stimulon (Cambridge Bioscience, Warchester, Mass., USA), adjuvants such as SAF-1 (Syntex), or bacterial DNA-based adjuvants such as ISS (Dynavax) or CpG (Corey Pharmaceuticals), and QS21 and 3-de Such as O-acetylated monophosphoryl lipid A (WO94 / 00153) or MF-59 (Chiron) or poly [di (carboxylatophenoxy) phosphazene], base adjuvant (Virus Research Institute), or Optivax (Vaxcel, Cytex) Block copolymer based adjuvant or Algammulin and Gamma I Adjuvants such as a combination of insulin-based adjuvants such as nulin (Anutech), incomplete Freund's adjuvant (IFA) or Gerbu preparation (Gerbu Biotechnik) can also be used. It should be understood that complete Freund's adjuvant (CFA) can also be used for non-human applications and for research purposes. "Vaccine composition" further includes excipients that are essentially non-toxic and non-therapeutic, such as water, saline, glycerol, ethanol, wetting or emulsifying pH buffer substances, preservatives, etc. And a diluent. Typically, vaccine compositions are prepared as injectables, either as liquid solutions or suspensions. Infusion can be subcutaneous, muscle, intravenous, intraperitoneal, intrathecal, intradermal. Other types of administration include transplantation, suppositories, ingestion, enteric application, inhalation, aerosolization or nasal spray or droplets. Solid forms suitable for dissolution on a liquid vehicle prior to injection or suspension in the liquid vehicle can also be prepared. The preparation can also be emulsified or encapsulated in a ribosome to enhance the adjuvant effect. The polypeptide can also be incorporated into an Immuno Stimulating Complex together with a saponin such as Quil A (ISCOMS). The vaccine composition comprises an effective amount of the active substance as well as any of the other above-mentioned ingredients. An “effective amount” of an active substance is effective to administer that amount to an individual as a single dose or as part of a series of doses in order to prevent or treat a disease or induce a desired effect. It means that. This amount depends on the health and physical condition of the individual to be treated, the taxonomic group of individuals to be treated (eg humans, non-human primates, primates, etc.), the ability of the individual's immune system to elicit an effective immune response. , Depending on the degree of protection desired, vaccine prescription, assessment of the treating physician, strains of infectious agents and other relevant factors. This amount is expected to fall within a relatively wide range that can be determined through routine trials. Usually this amount will be 0.01-1000 μg, more specifically 0.1-100 μg per dose. Additional dosage forms suitable for other modes of administration include oral dosage forms and suppositories. Dosage regimes can be single dose or multiple dose administration. The vaccine can be administered in combination with other immunomodulators.

  The invention is illustrated by the examples described below. These examples are for illustrative purposes only and are not to be construed as limiting or limiting the invention in any way.

Construction of PFPMT-MFα-E1-H6 shuttle vector A plasmid for Hansenula polymorpha transformation was constructed as follows. The pFPMT-MFα-E1-H6 shuttle vector was constructed in a multi-step procedure. First, the nucleic acid sequence CSN21 encoding the HCV E1s protein was cloned according to the CHH leader sequence (CHH = Carcinus maenas glycemic hormone), which was subsequently changed to the MFα leader sequence (MFα = Saccharomyces cereriae α-mating factor).

  First, a pUC18 derivative including CHH-E1-H6 units as an EcoRI / BamHI fragment was constructed by a seamless cloning method (Padgett, KA, and Sorge, JA, 1996). In addition, the E1s-H6-coding DNA fragment and the pCHH-Hiv-derived acceptor plasmid were obtained by PCR as described below.

（配列番号７）；
Ｅａｍ１１０４Ｉ部位には下線が付され、ドットは開裂部位をマークしている。太字で印刷された塩基は、プライマーＣＨＨ−ｌｉｎｋｓの塩基に対し相補的である。マークの付いていない塩基は、センス方向でＥ１の開始領域（１９２〜３２６）の中にアニールする。
−ＣＨＨＥ１−Ｒ：

（配列番号８）；
Ｅａｍ１１０４Ｉ部位には下線が付され、ドットは開裂部位をマークしている。太字で印刷された塩基は、プライマーＭＦ３０−ｒｅｃｈｔｓの塩基に対し相補的である。その後のクローニング手順に有用なＢａｍＨＩ部位を形成する塩基はイタリックで印刷されている。マークの付いていない塩基は、停止コドン及び停止コドンとＢａｍＨＩ部位との間の３つの塩基を含め、Ｅ１−Ｈ６単位の末端中にアンチセンス方向にアニールする。 Generation of an E1s-H6-coding DNA fragment. The plasmid PGEMTE1sH6 (SEQ ID NO: 6, FIG. 1) is encoded by PCR [encoding an HCV1bE1s type protein consisting of amino acids 192 to 326 of E1s extended with 6-His-residues (sequence) No. 5)] The E1-H6 DNA fragment was isolated. In addition, the following primers were used.
-CHHE1-F:

(SEQ ID NO: 7);
The Eam1104I site is underlined and the dots mark the cleavage site. The base printed in bold is complementary to the base of primer CHH-links. Unmarked bases anneal into the E1 start region (192-326) in the sense direction.
-CHHE1-R:

(SEQ ID NO: 8);
The Eam1104I site is underlined and the dots mark the cleavage site. The base printed in bold is complementary to the base of primer MF30-rechts. Bases that form a BamHI site useful for subsequent cloning procedures are printed in italics. The unmarked base anneals in the antisense direction into the end of the E1-H6 unit, including the stop codon and three bases between the stop codon and the BamHI site.

  The reaction mixture was configured as follows. 20 ng Eco311-linearized pGEMTE1sH6, each 0.2 μM primers CHHE1-F and CHHE1-R, dNTP ′S (at 0.2 μM each), 1 × buffer 2 (Expand Long Template PCR system; Boehringer: catalog number 1681 834), a total volume of 50 μL containing 2.5 u of polymerase mixture (Expand Long Template PCR system; Boehringer: catalog number 1681 834).

1. Using program 1 consisting of the following steps Denaturation: 5 minutes at 95 ° C. 10 cycles of denaturation at 95 ° C. for 30 seconds, annealing at 65 ° C. for 30 seconds, and elongation at 68 ° C. for 130 seconds.
3. Termination at 4 ° C.

50 μL of 10 × Buffer 2 (Expand Long Template PCR system; Boehringer; catalog number 1681 834), 40 μL H ₂ O and 5 μL [dATP, dGTP and dTTP (2 mM each); 10 mM 5-methyl dCTP] Amplification was performed according to a program consisting of the following steps.
1. Denaturation; 5 minutes at 95 ° C. 2. 5 cycles of denaturation at 95 ° C for 45 seconds, annealing at 65 ° C for 30 seconds, elongation at 68 ° C for 130 seconds.
3. Terminate at 4 ° C.

（配列番号１０）；
Ｅａｍ１１０４Ｉ部位には下線が付され、ドットは開裂部位をマークしている。太字で印刷された塩基は、プライマーＣＨＨＥ１−Ｆの塩基に対し相補的である。マークの付いていない塩基はアンチセンス方向でＣＨＨ配列の末端中にアニールする。
２．ＣＨＨ−Ｌｉｃｈｔｓ：

（配列番号１１）；
Ｅａｍ１１０４Ｉ部位には下線が付され、ドットは開裂部位をマークしている。太字で印刷された塩基は、プライマーＣＨＨＥ１−Ｒの塩基に対し相補的である。マークの付いていない塩基は、インサートから離れるような方向で、ｐＣＨＨ−ＨｉｒのクローニングされたＣＨＨ−Ｈｉｒｕｄｉｎ ＨＬ２０の後ろのｐＵＣ１８配列中にアニールする。 Generation of a receptor plasmid derived from pCHH-Hir A receptor plasmid was prepared by PCR from the pCHH-Hir plasmid (SEQ ID NO: 9; FIG. 2), except that the Hir coding sequence was not present in the PCR product. It is composed of an almost complete pCHH-Hir plasmid. The following primers were used for this PCR.
1. CHH-Links:

(SEQ ID NO: 10);
The Eam1104I site is underlined and the dots mark the cleavage site. The base printed in bold is complementary to the base of primer CHHE1-F. Unmarked bases anneal into the end of the CHH sequence in the antisense direction.
2. CHH-Lichts:

(SEQ ID NO: 11);
The Eam1104I site is underlined and the dots mark the cleavage site. The base printed in bold is complementary to the base of primer CHHE1-R. Unmarked bases anneal into the pUC18 sequence behind the cloned CHH-Hirudin HL20 of pCHH-Hir in a direction away from the insert.

  The reaction mixture was configured as follows. 20 ng Asp718I-linearized pCHH-Hir, each 0.2 μM primers CHH-links and MF30-rechts, dNTP (at 0.2 μM each), 1 × Buffer 2 (Expand Long Template PCR system; Boehringer catalog number 1681 834 ), A total volume of 50 μL containing 2.5 u of polymerase mixture (Expand Long Template PCR system; Boehringer: catalog number-1681834).

  The program as described above was used.

Then 5 μL of 10 × Buffer 2 (Expand Long Template PCR system; Boehringer; catalog number 1681 834), 40 μL H ₂ O and 5 μL [dATP, dGTP and dTTP (2 mM each); 10 mM 5-methyl dCTP] Amplification was performed according to the program as described above.

Generation of vector pCHHE1 The pCHH-Hir-derived acceptor plasmid and E1s-H6-coding DNA fragment generated by PCR as described above were obtained using a PCR product purification kit (Qiagen) according to the supplier's specifications. Purified. The purified fragment was then digested separately with Eam1104I. The E1s-H6 DNA fragment was then ligated into the pCHH-Hir derived receptor plasmid using T4 ligase (Boehringer) according to the supplier's specifications.

  In the ligation mixture Coli. XL-Gold cells were transformed and analyzed for plasmid DNA of multiple ampicillin resistant colonies by digestion with EcoRI and BamHI. A positive clone was selected and called pCHHE1.

Generation of vector pFPMT-CHH-E1H6 The EcoRI / BamHI fragment of pCHHE1k was ligated with vector pFPMT121 (SEQ ID NO: 12: FIG. 3) digested with EcoRI / BamHI. T4 ligase (Boehringer) was used according to the supplier's instructions. E. The ligation mixture was used to transform ColiDH5αF cells. Several transformants were analyzed on the restriction pattern of plasmid DNA, and a positive clone was retained, which was designated pFPMT-CHH-E1H6 (SEQ ID NO: 13: FIG. 4).

Generation of pFMPT-MFα-E1-H6 Finally, shuttle vector pFMPT-MFα-E1-H6 was generated by ligation of the following three fragments.
1. 6.961 kb EcoRI / BamHI digested pFPMT121 (SEQ ID NO: 12: FIG. 3)
2. 2. an EcoRI / HindIII fragment of pUC18-MFa (SEQ ID NO: 62: FIG. 36) of 0.245 (kb); 0.442 kb HindIII / BamHI fragment of the 0.454 kb PCR product derived from pFPMT-CHH-E1H6.

（配列番号１４）及び
２．プライマーＥ１ｂａｃｋ−Ｂａｍ

（配列番号１５） By PCR using the following primers, fragment No. A 0.454 kb PCR product generating 3 was obtained.
1. Primer MFa-E1f-Hi:

(SEQ ID NO: 14) and 2. Primer E1back-Bam

(SEQ ID NO: 15)

The reaction mixture was configured as follows. Reaction mixture volume 50 μL, pFPMT-CHH-E1-H6 (EcoRI linearization; 15 ng / μL), 0.5 μL; primer MFa-E1f-Hi (50 μM), 0.25 μL; primer E1back-Bam (50 μM); 25 μL; dNTPs (all at 2 mM), 5 μL; DMSO, 5 μL; H ₂ O, 33.5 μL; Expand Long Template PCR system (Boehringer Mannheim; catalog number 1681 834) buffer 2 (concentrated 10-fold) 5 μL; Expand Long Template PCR system polymerase mixture (1 U / μL), 0.5 μL.

A PCR program consisting of the following steps was used.
1. Denaturation: 5 minutes at 95 ° C. 29 cycles of denaturation at 95 ° C for 45 seconds, annealing at 55 ° C for 45 seconds, and elongation at 68 ° C for 40 seconds.
3. Termination at 4 ° C.

  Based on the primers used, the resulting 0.454 kb PCR product contained an E1 (192-326) codon followed by 6 histidine codons and a “taa” stop codon, which was upstream Flanked by 22 3 ′ terminal base pairs of MFα prepro SA (including cloning-related HindIII sites plus 6 base pair overhangs) and downstream (cloning related) by BamHI sites and 6 base pair overhangs It was.

  For the ligation reaction, T4 DNA ligase (Boehringer Mannheim) was used according to the supplier's conditions (sample volume 20 μL).

  E. E. coli HB101 cells were transformed with the ligation mixture and positive clones were retained after restriction analysis of plasmids isolated from multiple transformants. A positive plasmid was selected and called pFPMT-MFα-E1-H6 (SEQ ID NO: 16, FIG. 5).

Construction of pFPMT-CL-E1-H6 shuttle vector A prosmid for transformation of Hansenula polymorpha was constructed as follows. The pFPMT-CL-E1-H6 shuttle vector was constructed in 3 steps starting from pFPMT-MFα-E1-H6 (SEQ ID NO: 16, FIG. 5).

  In the first step, the MFα-E1-H6 reading frame of pFPMT-MFα was subcloned into the pUC18 vector. Therefore, the 1.798 kb SalI / BamHI fragment of pFPMT-MFα-E1-H6 (including FMD promoter plus MFα-E1 = H6) was used to obtain the SalI / Ligated to the BamHI vector fragment. As a result, the plasmid (SEQ ID NO: 17) depicted in FIG. 6 was obtained, and this was designated as pMa12-1 (pUC18-FMD-MFα-E1-H6). E. The ligation mixture was used to transform E. coli DH5αF 'cells. Multiple ampicillin resistant colonies were picked and analyzed by restriction enzyme digestion of plasmid DNA isolated from the picked clones. Positive clones were further analyzed by determining the DNA sequence of the MFα-E1-H6 coding sequence. The correct clone was used for PCR-induced mutagenesis to replace the MFα prepro sequence with a codon of the avian lysozyme presequence (“CL”: corresponding to amino acids 1-18 of avian lysozyme; SEQ ID NO: 1). The principle of the applied PCR-induced mutagenesis method is based on amplification of the entire plasmid with the desired modification at the 5 'end of the primer. In the downstream step, the end of the linear PCR product is modified prior to self-ligation, resulting in the desired altered plasmid.

（配列番号１８）
２．プライマー ＣＬ ｈｅｒ ｎｅｕ；

（配列番号１９） The following primers were used for the PCR reaction.
1. Primer CL Hin;

(SEQ ID NO: 18)
2. Primer CL her neu;

(SEQ ID NO: 19)

  The underlined 5 'region of the primer contains about half the codon of the avian lysozyme presequence. Primer CLherneu includes a SpeI restriction site (italics). The underlined region of the primer is the codon for amino acid residues 192-199 of E1 (CL hin) or “atg” beyond the EcoRI site to position-19 of the FMD promoter (counted from the EcoRI site). Anneal with the start codon. The primer amplifies the complete pMa12-1, thus replacing the codons of the MFa prepro sequence with the codons of the avian lysozyme pre sequence.

The reaction mixture was composed as follows; pUC18-FMD-MFα-E1-H6 (pMa12-1; 1.3 ng / μL), 1 μL; primer CL hin (100 μM), 2 μL; primer CL her neu ( 100 μM), 2 μL; dNTP (all 2.5 mM), 8 μL; H ₂ O, 76 μL; Expand Long Template PCR system (Boehringer; catalog number 1681 834) Buffer 2 (concentrated 10-fold), 10 μL; Expand Long Template PCR System Polymerase mixture (1 U / μL), 0.5 μL.

A PCR program consisting of the following steps was used;
1. Denaturation: 15 minutes at 95 ° C. Denaturation at 95 ° C. for 30 seconds, annealing at 60 ° C. for 1 minute, and extension at 72 ° C. for 1 minute for 35 cycles.
3. Termination at 4 ° C.

The resulting PCR product was examined by agarose gel electrophoresis for its proper size (3.5 kb). The 3′-A overhanging form of the PCR product was then removed by T4 polymerase reaction, resulting in blunt ends with 3′- and 5′-OH groups. Therefore, the PCR product was treated with T4 polymerase (Boehringer; 1 U / μL). That is, 1 μL T4 polymerase and 4 μL dNTP (all at 2.5 mM) were added to the remaining 95 μL PCR reaction mixture. Samples were incubated for 20 minutes at 37 ° C. The DNA was then precipitated with ethanol and taken up in 16 μL H ₂ O.

  5'-phosphate was then added to the blunt end PCR product by a kinase reaction. Therefore, 1 μL of T4 polynucleotide kinase (Boehringer; 1 U / μL), 2 μL of 10-fold concentrated T4 polynucleotide kinase reaction buffer (Boehringer) and 1 μL of ATP (10 mM) are added to 16 μL of blunt-end PCR product. did. Samples were incubated at 37 ° C. for 30 minutes.

  DNA was then applied on a 1% agarose gel and the appropriate product band was isolated using a gel extraction kit (Qiagen) according to the supplier's conditions. 50 ng of purified product was then self-ligated using T4 ligase (Boehringer) according to the supplier's conditions. After incubating at 16 ° C. for 72 hours, the DNA in the ligation mixture was precipitated with ethanol and dissolved in 20 μL of water.

  Thereafter, E.E. E. coli DH5α-F ′ cells were transformed with 10 μL of the ligated sample. The plasmid DNA of multiple ampinline resistant clones was examined using restriction enzyme digestion. A positive clone was reserved and designated p27d-3 (pUC18-FMD-CL-E1-H6, SEQ ID NO: 20, FIG. 7). The CL-E1-H6 open reading frame was then confirmed by DNA sequencing.

In the final step, a pFPMTM-CL-E1-H6 shuttle vector was constructed as described below. A 0.486 kb EcoRI / BamHI fragment of p27d-3 (which includes CL-E1 (192-326) -H6) was ligated with pFPMT121 (SEQ ID NO: 12, FIG. 3) digested with EcoRI / BamHI. T4 ligase (Boehringer) was used for the reaction according to the supplier's recommendations. DNA in the ligated sample was precipitated with ethanol and dissolved in 10 μl of H ₂ O. Using 10 μL of the ligated sample, E. coli DH5αF ′ cells were transformed and plasmid DNA of multiple ampicillin resistant colonies was analyzed by digestion with EcoRI and BamHI. Plasmid clone p37-5 (pFPMT-CL-E1-H6: SEQ ID NO: 21, FIG. 8) showed the desired fragment sizes of 0.486 kb and 6.961 kb. Sequencing confirmed the proper sequence of CL37-E1-H6 of p37-5.

Construction of pFPMT-MFα-E2-H6 and pMPT-MFα-E2-H6 shuttle vectors Hansenula polymorpha plasmids were constructed as follows. The DNA sequence encoding MFα-E2s (HCV E2 amino acids 384-673) -VIEGR-His6 (SEQ ID NO: 5) was isolated from plasmid pSP72E2H6 (SEQ ID NO: 22, FIG. 9) as a 1.331 kb EcoRI / BglII fragment. . This fragment was either pFPMT121 (SEQ ID NO: 12, FIG. C + 2) or pMPT121 (SEQ ID NO: 23, FIG. 10) digested with EcoRI / BglII using T4 DNA ligase (Boehringer Mannheim) according to the supplier's recommendations. It was connected with either of. E. E. coli was transformed, and plasmid DNAs isolated from different transformants by restriction enzyme digestion were examined. Then, positive clones were retained, and the resulting shuttle vector was transferred to pFPMT-MFα-E2-H6 (SEQ ID NO: 22, FIG. 11) and pMPT-MFα-E2-H6 (SEQ ID NO: 23, FIG. 12).

Construction of pFPMT-CL-E2-H6 shuttle vector The shuttle vector pFPMT-CL-E2-H6 was assembled in a three step procedure. The E2 coding sequence was cloned behind the signal sequence of the Schwannomycines occidentalis α-amylase. This was done by a seamless cloning method (Padgett, KA, and Sorge JA, 1996).

（配列番号２６）；ＥａｍＩ１０４Ｉ部位には下線が付され、ドットは酵素の開裂部位をマークしている。Ｓ．ｏｃｃｉｄｅｎｔａｌｉｓシグナル配列の前後のコドンは太字で印刷されている；マークの付いていない塩基はＥ２のコドン（ＨＣＶ Ｅ２のアミノ酸３８４〜３９０）とアニールする。
−プライマーＭＦ３０Ｅ２／Ｒ

（配列番号２７）；ＥａｍＩ１０４Ｉ部位には下線が付され、ドットは酵素の開裂部位をマークしている。太字で印刷された塩基は、プライマーＭＦ３０−Ｒｅｃｈｔｓの塩基に対し相補的である。（以下参照）、構築物に導入されるべきＢａｍＨＩ部位は、イタリック体で印刷されている。マークされていない配列は停止コドン及びＥ２（３８４−６７３）−ＶＩＥＲ−Ｈ６（配列番号５）の６つの末端Ｈｉｓコドンとアニールする。 Generation of E2s-H6 Coding DNA Fragment First, a DNA sequence encoding E2-H6 (linker peptide “VIEGR” and 6His residues, amino acids 384-673 of HCV E2 extended with SEQ ID NO: 5) is transformed into the pSP72E2H6 plasmid (sequence No. 24, FIG. 11) was amplified by PCR. The primers used were designated MF30E2 / F and MF30E / R and have the following sequences:
-Primer MFE2 / F;

(SEQ ID NO: 26); the EamI104I site is underlined and the dots mark the cleavage site of the enzyme. S. The codons before and after the ocidentalis signal sequence are printed in bold; unmarked bases anneal to the E2 codon (amino acids 384-390 of HCV E2).
-Primer MF30E2 / R

(SEQ ID NO: 27); the EamI104I site is underlined and the dots mark the cleavage site of the enzyme. The base printed in bold is complementary to the base of primer MF30-Rechts. (See below) BamHI sites to be introduced into the construct are printed in italics. The unmarked sequence anneals with a stop codon and the six terminal His codons of E2 (384-673) -VIER-H6 (SEQ ID NO: 5).

  The reaction mixture was structured as follows: 20 ng pSP72E2H6 1.33 kb EcoRI / BglII fragment each 0.2 μM primer MF30E2 / F and MF30E2 / R, dNTP (each at 0.2 μM), 1 × buffer 2 (Expand Long Template PCR system; Boehringer, catalog number 1681 834), a total volume of 50 μL containing 2,5 U of polymerase mixture (Expand Long Template PCR system; Boehringer, catalog number 1681 834).

Using program 3 consisting of the following steps: Denaturation; 5 minutes at 95 ° C. 2. 10 cycles of denaturation at 95 ° C. for 30 seconds, annealing at 65 ° C. for 30 seconds, and extension at 68 ° C. for 1 minute.
3. Termination at 4 ° C.

Then 10 μL of 10 × Buffer 2 (Expand Long Template PCR system; Boehringer, catalog number 1681 834), 40 μL H ₂ O and 5 μL [dATP, dGTP and dTTP (2 mM each); 10 mM, 5-methyl-dCTP ] Was added to the sample from PCR program 3 and continued with program 4 consisting of the following steps.
1. Denaturation; 5 minutes at 95 ° C. 2. Denaturation at 95 ° C for 45 seconds, annealing at 65 ° C for 30 seconds, extension at 68 ° C for 1 minute, 5 cycles.
3. Terminate at 4 ° C.

（配列番号２９；ＥａｍＩ１０４Ｉ部位には下線が付され、ドットは酵素の開裂部位をマークしている。太字で印刷された「ｃｃｔ」は、プライマーＭＦ３０Ｅ２／Ｆの太字で印刷された「ａｇｇ」に対し相補的である。（以上参照）；マークの付いていない及び太字印刷された塩基は、ｐＭＦ３０内のＳ．ｏｃｃｉｄｅｎｔａｌｉｓのα−アミラーゼのコドンの２６の末端塩基とアニールする）−プライマーＭＦ３Ｕ−Ｒｅｃｈｔｓ；

（配列番号２９；ＥａｍＩ１０４Ｉ部位には下線が付され、ドットは酵素の開裂部位をマークしている。太字で印刷された「ｃｔｇ」は、プライマーＭＦ３０Ｅ２／Ｒの太字印刷された「ｃａｇ」に対し相補的である。（以上参照）マークの付いていない塩基はｐＭＦ３０中のＳ．ｏｃｃｉｄｅｎｔａｌｉｓのα−アミラーゼの停止コドンの下流側のｐＵＣ１８配列とアニールする。） Generation of a receptor plasmid derived from PMF30 The second fragment is derived from plasmid pMF30 (SEQ ID NO: 28, FIG. 13) and The complete pMF30 plasmid, except for the codons of the mature Occidentalis α-amylase, modifications related to cloning were introduced by primer design. The following primer sets were used.
-Primer MF30-Links;

(SEQ ID NO: 29; the EamI104I site is underlined and the dot marks the cleavage site of the enzyme. The “cct” printed in bold is the “agg” printed in bold in the primer MF30E2 / F. Complementary to (see above); unmarked and bold printed bases anneal to the 26 terminal bases of the codon of S. occidentalis α-amylase in pMF30) —Primer MF3U-Rechts ;

(SEQ ID NO: 29; the EamI104I site is underlined and the dot marks the cleavage site of the enzyme. The “ctg” printed in bold is the “cag” printed in bold for the primer MF30E2 / R. Complementary (see above) Unmarked bases anneal to the pUC18 sequence downstream of the stop codon of S. occidentalis α-amylase in pMF30.

  The reaction mixture was structured as follows: 20 ng -BglII-linearized pMF30, 0.2 μM each of primers MF30-Links and MF30-Rechts, dNTP (each at 0.2 μM), 1 × buffer 1 (Expand Long Template PCR system; Boehringer: catalog number 1681 834), total volume of 50 μL containing 2.5 U of polymerase mixture (Expand Long Template PCR system; Boehringer: catalog number 1681 834). The same PCR program (programs 3 and 4) as described above was used with the exception of the extension time extended from 1 minute to 4 minutes in both programs.

Generation of vector pAMY-E2 The pMF30-derived receptor plasmid and E2s-H6 coding DNA fragment obtained by PCR were examined on the basis of their respective sizes by gel electrophoresis on a 1% agarose gel. The PCR product was purified with a PCR product purification kit (Qiagen) according to the supplier's instructions. The purified fragment was then digested separately with Eam11004I. Ligation of the E2s-H6 fragment with the pMF30 derived receptor plasmid was performed using T4 ligase (Boehringer) according to the supplier's recommendations. Using the ligation mixture, E.I. E. coli DH5αF ′ cells were transformed and the plasmid DNA of multiple clones was analyzed by EcoRI / BamHI digestion. A positive clone was selected and the plasmid was further designated pAMY-E2 and used for further modifications as described below.

（配列番号３０）及び
−プライマー ＣＬ２ ｈｅｒ：

（配列番号３１） Generation of vector pUC18-CL-E2-H6 pAMY-E2 was subjected to PCR-induced mutagenesis in order to replace the codon of the α-amylase signal sequence with the codon of the avian lysozyme presequence. This is further referred to as “CL” and corresponds to the first 18 amino acids of the avian lysozyme ORF (SEQ ID NO: 1). The following primers were used for this mutagenesis:
-Primer CL Hin:

(SEQ ID NO: 30) and -Primer CL2 her:

(SEQ ID NO: 31)

  The underlined 5 'region of the primer contains about half the DNA sequence of the avian lysozyme presequence. Primer CL2 her includes SpeI (italic) and EcoRI (italic, double underlined) restriction sites. The underlined region of the primer is the “atg” from the codon of amino acid residues 384-392 of E2 (CL2 hin) or from the EcoRI site to position-19 of the FMD promoter (counted from the EcoRI site). Anneal with the start codon. The primer amplifies the complete pAMY-E2 vector, thus replacing the codon of the α-amylase signal sequence with the codon of the avian lysozyme presequence.

The PCR reaction was performed according to the following program:
1. Denaturation; 15 minutes at 95 ° C.
2. Denaturation at 95 ° C. for 30 seconds, annealing at 60 ° C. for 1 minute, and extension at 72 ° C. for 1 minute for 35 cycles.
3. Termination at 4 ° C.

The following reaction mixture was used: pAMY-E2 (1 ng / μL), 1 μL; Primer CL Hin (100 μM), 2 μL; Primer CL2 her (100 μM), 2 μL; dNTP (all 2.5 mM), 8 μL; H ₂ O Expand Long Template PCR system (Boehringer); Cat No 1681 834 buffer 2 (concentrated 10-fold), 10 μL: Expand Long Template PCR system polymerase mixture (1 U / μL), 0.75 μL.

  The resulting PCR product was examined by gel electrophoresis on a 1% agarose gel. Prior to ligation, the PCR fragment was modified as follows. The 3'-A overhang was removed by T4 polymerase, resulting in blunt ends with 3'- and 5'-OH groups. Furthermore, 1 μL of T4 polymerase (Boehringer; 1 U / μL) was added to 4 μL dNTPs (all at 2.5 mM) to the remaining 95 μL of the PCR reaction mixture. Samples were incubated for 20 minutes at 37 ° C. The DNA was then precipitated with ethanol and dissolved in 16 μL of deionized water. This was followed by 1 μL of T4 polynucleotide kinase (Boehringer); 1 U / μL for 16 μL of the dissolved blunt-ended PCR product that was kinased to add 5′-phosphate to the blunt-ended PCR product; 2 μL of 10 × concentrated T4 polynucleotide kinase reaction buffer (Boehringer) and 1 μL of ATP (10 mM) were added. Samples were incubated at 37 ° C. for 30 minutes.

  The kinase treated sample was then separated on a 1% agarose gel. DNA was extracted from agarose slices using a gel extraction kit (Qiagen) according to the supplier's recommendations. 50 ng of purified product was then self-ligated using T4 ligase (Boehringer) according to the supplier's conditions. After incubation at 16 ° C. for 16 hours, the DNA in the ligation mixture was precipitated with ethanol and dissolved in 20 μL of water (ligation sample).

  E. E. coli DH5α-F ′ cells were transformed with 10 μL of ligated sample. Multiple ampicillin resistant clones were characterized using restriction analysis of isolated plasmid DNA. The positive clone was designated PUC18-CL-E2-H6 and was used for further modifications as described below.

Generation of shuttle vector pFPMT-CL-E2-H6 (including CL-E2 (384-673) -VIEGR-H6) A 0.966 kb EcoRI / BamHI fragment was isolated from pUC18-CL-E2-H6 and EcoRI Ligated into pFPMT121 (SEQ ID NO: 12, FIG. 3) digested with / Bam HI. For the reaction, T4 ligase (Boehringer) was used according to the supplier's conditions. Ligated samples were precipitated with ethanol and dissolved in 10 μL of water. Using this, E.I. E. coli DH5αF ′ cells were transformed, positive clones were retained after restriction analysis, and each plasmid was designated pFPMT-CL-E2-H6 (SEQ ID NO: 32, FIG. 4).

Construction of pFPMT-CL-K-H6-E1 shuttle vector Construction of the shuttle vector consists of two steps.

（配列番号３７）
−プライマー Ｈ６ＫＲＫ ｈｅｒ ｎｅｕ：

（配列番号３８）
（付加的なコドンを提供する塩基には下線が付されている）。 In the first step, the pUC18-FMD-CL-H6-K-E1-H6 construct was constructed by site-directed mutagenesis. pUC18-FMD-CL-E1-H6 was used as a template (SEQ ID NO: 20; FIG. 7). The following primers were used.
-Primer H6K hin neu:

(SEQ ID NO: 37)
-Primer H6KRK her neu:

(SEQ ID NO: 38)
(Bases that provide additional codons are underlined).

The PCR reaction mixture was structured as follows: pUC18-FMD-CL-E1-H6 (2 ng / μL), 1 μL; primer H6K hin neu (100 μM)
2 μL: Primer H6KRK her neu (100 μM), 2 μL; dNTP (2.5 mM each), 8 μL; H ₂ O, 76 μL; Expand Long Template PCR system (Boehringer; Cat No 1681 834) buffer 2 (concentrate 10 times) 10 μL: Expand Long Template PCR system polymerase mixture (1 U / μL), 0.75 μL

The PCR program used consisted of the following steps:
-Denaturation: 15 minutes at 95 ° C-35 cycles of denaturation at 95 ° C for 30 seconds, annealing at 60 ° C for 1 minute, extension at 72 ° C for 5 minutes.
-Terminate at 4 ° C.

  An aliquot of the PCR sample was analyzed on a 1% agarose gel to check its size, which was correct (˜4.2 kb).

Subsequently, the 3′-A overhang from the PCR product was removed by a T4 polymerase reaction, resulting in blunt ends with 3′- and 5′-OH groups. Therefore, 1 μL of T4 polymerase (Boehringer; 1 U / μL) and 4 μL of dNTP (2.5 mM each) were added to the remaining 95 μL of the PCR reaction. Samples were incubated at 37 ° C. for 20 minutes. Thereafter, the DNA in the sample was precipitated with ethanol and dissolved in 16 μL of H ₂ O.

  Subsequently, 5'-phosphate was added to the blunt end PCR product by a kinase reaction. Thus, for 16 μL of lysed blunt end PCR product, 1 μL of T4 polynucleotide kinase (Boehringer); 1 U / μL), 2 μL of 10 × concentrated T4 polynucleotide kinase reaction buffer (Boehringer) and 1 μL of ATP (10 mM) ) Was added. Samples were incubated at 37 ° C. for 30 minutes.

  Samples were then applied on a 1% agarose gel and the appropriate product band was isolated using a gel extraction kit (Qiagen) according to the supplier's conditions. 50 ng of purified product was then self-ligated using T4 ligase (Boehringer) according to the supplier's recommendations. After incubation at 16 ° C. for 72 hours, the DNA in the ligation mixture was precipitated with ethanol and dissolved in 10 μL of water.

  E. E. coli DH5α-F ′ cells were transformed with 5 μL of the ligated sample. Multiple ampicillin resistant colonies were analyzed by restriction enzyme digestion, positive clones were retained and the corresponding plasmid was designated as follows: pUC18-FMD-CL-H6-E1-K-H6 (SEQ ID NO: 39, FIG. 17) ).

  In the second step, a transfer vector was constructed by two-fragment ligation. In the following, a construction fragment with a BclI sticky end was involved. Since BalI can only cleave that site on unmethylated DNA, The E. coli dam-strain was transformed with the involved plasmids pUC18-FMD-CL-H6-K-E1-H6 (SEQ ID NO: 39, FIG. 17) and pFPMT-CL-E1 (SEQ ID NO: 36, FIG. 16). From each transformation, ampicillin resistant colonies were picked and grown in liquid culture and unmethylated plasmid DNA was prepared for further use. 1.273 kb BclI / Hind III fragment of unmethylated plasmid pUC18-FMD-CL-H6-K-E1-H6 (including FMD promoter, CL-H6-K unit codon and E1 start), and Prepare a 6.057 kb BclI / HindIII fragment of plasmid pFPMT-CL-E1 (including the missing portion of the E1 reading frame starting from the BclI site without the C-terminal His tag as well as the elements present in pFPMT121 except for the FMD promoter) And ligated for 72 hours at 16 ° C. using T4 ligase (Boehringer) in a total volume of 20 μL according to the supplier's specifications. The mixture was then placed on a piece of nitrocellulose membrane suspended on sterile deionized water for the purpose of desalting the ligation mixture (30 minutes incubation at room temperature). E. coli by electrophoresis on 5 μL of desalted sample. E. coli TOP10 cells were transformed. The resulting plasmid DNA of multiple ampicillin resistant colonies was analyzed by restriction enzyme digestion. A positive clone was withheld and was designated pFPMT-CL-H6-K-E1 (SEQ ID NO: 40, FIG. 18).

Transformation of Hansenula polymorpha and selection of transformants polymorpha strain RB11 was transformed (basically (clave) using modifications with different parent shuttle vectors as described in Examples 1-5 (Rogenkamp, R. et al., 1986). (PEG-mediated DNA uptake protocol as described in (Klebe, RJ) et al., 1983)). For each transformation, 72 uracil prototrophic colonies were selected and used for strain generation by the following procedure. For each colony, 2 mL of liquid culture was inoculated and grown in selective medium (YNB / glucose, Difco) in a test tube (37 ° C .; 160 rpm; angle 45 ° C.) for 48 hours. This step is defined as the first passage step. A 150 μL aliquot of the first passage culture was used to inoculate 2 mL of fresh YNB / glucose medium. Again, the culture was incubated as described above (second passage step). Together, the passage process was performed 8 times. A culture aliquot after the third and eighth passage steps was used to inoculate 2 mL of non-selective YPD medium (Difco). After a 48 hour incubation at 37 ° C. (160 rpm; angle 45 ° C .; so-called first stabilization step), a 150 μL aliquot of these YPD cultures was used to prepare a fresh 2 mL YPD culture incubated as described above. Inoculated (second stabilization step). An aliquot of the second stabilization step culture was then streaked on a plate containing selective YNB / agar. These plates were incubated for 4 hours until macroscopic colonies became visible. A distinct single colony for each segregation was defined as the strain and used for further expression analysis.

  Expression analysis was performed on small scale shake flask cultures. Colonies were picked from the YNB / agar plates described above, inoculated into 2 mL YPD and incubated for 48 hours as described above. This 2 mL aliquot was used as a seed culture for a 2 mL shake flask culture. YP glycerol (1%) was used as the medium, and the shake flask was incubated on a rotary shaker (200 rpm, 37 ° C.). After 48 hours of growth, 1% MeOH was added to the culture for induction of the expression cassette. At different time intervals, 1 mL aliquots of cell pellets were collected and stored at −20 ° C. until further analysis. Specific protein expression was analyzed by SDS-PAGE / Western blotting. Therefore, the cell pellet was solubilized in sample buffer (Tris HCl-SDS) and incubated at 95 ° C. for more than 15 minutes.

  Proteins were separated on a 15% polyacryl-amide gel and blotted onto a nitrocellulose membrane (wet blot; bicarbonate buffer). Blots were developed using specific mouse anti-E1 (IGH201) or mouse anti-E2 (IGH216 described by Maertens et al., In WO 96/04385) as the first antibody, and rabbit anti-mouse-AP was used as the second antibody. Used as an antibody. Staining was performed with NBT-BCIP.

  Positive strains were withheld for further investigation.

  Five of these positive clones were used in shake flask expression experiments. Colonies of each strain were picked from the YNB plate and used to inoculate 2 mL of YPD. These cultures were incubated as described above. This cell suspension was used to inoculate a second seed culture of 100 mL YPD medium in a 500 mL shake flask. The shake flask was cultured on a rotary shaker at 37 ° C. and 200 rpm for 48 hours. A 25 mL aliquot of this seed culture was used to inoculate 250 mL of YP glycerol (1%) medium and incubated in a baffled 2 l shake flask under the conditions described above. 48 hours after inoculation, 1% MeOH (promoter induction) was added and the shake flasks were further incubated under the conditions described above. 24 hours after induction, the experiment was stopped and the cell pellet was collected by centrifugation. The expression levels of 5 different clones were analyzed by SDS-PAGE / Western blotting (conditions as described above). A titration series of each clone on the gel was loaded and the most productive strain was selected for further fermentation and purification trials.

  Surprisingly, Pichia pastoris (Gellissen), G.M. 2000) which is a yeast strain having a relationship close to 2000). polymorpha is capable of expressing HCV protein with a sugar moiety that is essentially free of hyperglycosylation and thus with a size of sugar comparable to that of HCV virus-like particles expressed by mammalian cells infected with HCV recombinant vaccinia virus. it can.

  Hansenula polymorpha strain RB11 was established in UCL Mycotheque (MUCL) Louvain Catholic University Mycology Laboratory (Place Croix du Sud 3bte 6, B-1348 Louvain-la-Neuve, Belgium) in April 2002 under the conditions of the Budapest Treaty. Deposited on the day and has MUCL deposit number MUCL43805.

Construction of pSY1aMFElsH6a vector A cerevisiae expression plasmid was constructed as follows. The E1 coding sequence was isolated as an NsI1 / Eco52I fragment from pGEMT-E1sH6 (SEQ ID NO: 6, FIG. 1), blunt-ended (using T4 DNA polymerase) and using T4 DNA ligase (Boehringer) according to the supplier's specifications. In the pYlG5 vector (SEQ ID NO: 41, FIG. 19). Cloning was such that the E1s-H6 coding fragment was joined directly and in frame with the aMF coding sequence. The ligation mixture is obtained from E. coli. E. coli DH5αF ′ cells were transformed. Subsequently, plasmid DNA of multiple ampicillin resistant clones was analyzed by restriction digestion, positive clones were withheld and designated pYIG5E1H6 (ICCG3470; SEQ ID NO: 42, FIG. 20).

  The expression cassette (containing the E1s coding region with His-tag and the αMF-sequence) was digested with BamHI. coli / S. cerevisiae pSY1 shuttle vector (SEQ ID NO: 21, FIG. 43) was transferred as a BamHI fragment (2790 bp) of pYIG5E1H6. Ligation was performed with T4 DNA ligase (Boehringer) according to the supplier's conditions. The ligation mixture is obtained from E. coli. E. coli DH5αF 'cells were transformed and plasmid DNA of multiple ampicillin resistant colonies was analyzed by restriction enzyme digestion. A positive clone was reserved and designated pSY1aMFE1sH6a (ICCG3479; SEQ ID NO: 44, FIG. 22).

Construction of pSYYIGSE2H6 vector cerevisiae expression plasmid pSYYIGSE2H6 was constructed as follows. The E2 coding sequence was isolated as a SalI / KpnI fragment from pBSK-E2sH6 (SEQ ID NO: 45, FIG. 23), which was blunted (using T4 DNA polymerase) and then T4 DNA ligase (Boehringer) according to the supplier's specifications. Was cloned into the pYlG5 vector (SEQ ID NO: 41, FIG. 19). Cloning was such that the E2-H6 coding fragment was joined directly and in-frame with the aMF coding sequence. The ligation mixture is then treated with E. coli. E. coli DH5αF ′ cells were transformed, plasmid DNA of multiple ampicillin resistant clones was analyzed by restriction digest, positive clones were retained and designated pYIG5HCCL-22aH6 (ICCG2424; SEQ ID NO: 46, FIG. 24).

  An expression cassette (containing the E2 (384-673) coding region with His-tag and αMF-sequence) was transformed into E. coli opened with BamHI. coli / S. cerevisiae pSY1 shuttle vector (SEQ ID NO: 43, FIG. 21) was transferred as a BamHI fragment (3281 bp) of pYIG5HCCL-22aH6. Ligation was performed with T4 DNA ligase (Boehringer) according to the supplier's conditions. The ligation mixture is obtained from E. coli. E. coli DH5αF 'cells were transformed and plasmid DNA of multiple ampicillin resistant colonies was analyzed by restriction enzyme digestion. A restriction positive clone was withheld and called pSYIGSE2H6 (ICCG2466; SEQ ID NO: 47, FIG. 25).

Construction of pSY1Y1G7E1s vector cerevisiae expression plasmid pSY1YIG7E1s was constructed as follows. The E1 coding sequence was isolated as an NsI1 / Eco52I fragment from pGEMT-E1s (SEQ ID NO: 6, FIG. 1), blunt-ended, and pYlG7 vector (SEQ ID NO: 48) using T4 DNA ligase (Boehringer) according to the supplier's specifications. In FIG. 26), cloning was performed. Cloning was such that the E1 coding fragment was joined directly and in frame with the aMF coding sequence. The ligation mixture is obtained from E. coli. E. coli DH5αF ′ cells were transformed, plasmid DNA of multiple ampicillin resistant clones was analyzed by restriction digestion, positive clones were retained and designated pYIG7E1 (SEQ ID NO: 49, FIG. 27).

  An expression cassette (containing the E1 (192-326) coding region and CL leader sequence) was digested with BamHI. coli / S. cerevisiae pSY1 shuttle vector (SEQ ID NO: 43, FIG. 21) was transferred as a BamHI fragment (2790 bp) of pYIG7E1. Ligation was performed with T4 DNA ligase (Boehringer) according to the supplier's conditions. The ligation mixture is obtained from E. coli. E. coli DH5αF 'cells were transformed and plasmid DNA of multiple ampicillin resistant colonies was analyzed by restriction enzyme digestion. A positive clone was withheld and designated pSY1YIG7E1s (SEQ ID NO: 50, FIG. 28).

Transformation of Saccharomyces cerevisiae and selection of transformants A mutant screen was set up to overcome the hyperglycosylation problem often reported for proteins overexpressed in Saccharomyces cerevisiae. This screen was based on Barrow's method (Barrow L. et al., 1991) selecting spontaneous recessive orthovanadate resistant mutants. Initial strain selection was performed based on the glycosylation pattern of invertase as seen after native gel electrophoresis. Strains with reduced glycosylation capacity were withheld for further recombinant protein expression experiments, which predominated over strain IYCC155. The nature of the mutation was not studied further.

  The glycosylation deficient strain IYCC155 was transformed with the plasmid as described in Examples 7-9 by the lithium acetate method, essentially as described by EIble, R. 1992. Multiple Ura complementary strains were picked from selective YNB + 2% agar plates (Difco) and used to inoculate 2 ml of YNB + 2% glucose. These cultures were incubated at 37 ° C. for 72 hours at 200 rpm on an orbital shaker and developed by E1 specific mouse monoclonal antibody (IGH201) by Western blot for expression of culture supernatant and intracellular fractions. Minutes were analyzed. Highly productive clones were withheld for further experiments.

  S. used here. Expression of the protein within the Cerivisiae glycosylation-deficient mutation is This is hampered by the suboptimal growth characteristics of such strains, which leads to low biomass yields and thus lower yields of the desired protein compared to cerevisiae strains. The yield of the desired protein was still substantially higher than in mammalian cells.

Construction of pPICZalphaD'ElsH6 and pPICZalphaE'E1sH6 vectors Starting from the pPICZalphaA vector (Invitrogen; SEQ ID NO: 51, Figure 29), the shuttle vector pPICZalphaE'E1sH6 was constructed. In the first step, the vector was adapted to allow cloning of the E1 coding sequence immediately after the cleavage site of the KEX2 or STE13 treatment protease, respectively. Therefore, pPIC ZalphaA was digested with XhoI and NotI. The digests were separated on a 1% agarose gel and the 3519 kb fragment (most of the vector) was isolated and purified using a gel extraction kit (Qiagen). This FG is then ligated with T4 polymerase (Boehringer) in the presence of a specific oligonucleotide according to the supplier's conditions to give pPIC Zalpha D ′ (SEQ ID NO: 52, FIG. 30) or pPIC Zalpha E ′ (SEQ ID NO: 53, FIG. 31).

（配列番号５４）及び

（配列番号５５）
これは、アニーリングの後、以下のリンカーオリゴヌクレオチドを生成する：

（配列番号５４）

（配列番号５５）
− ｐＰＩＣ ＺａｌｐｈａＥを構築するためには、

（配列番号５６）

（配列番号５７）
これは、アニーリングの後、以下のリンカーオリゴヌクレオチドを生成する：

（配列番号５６）

（配列番号５７） The following oligonucleotides were used.
-To construct pPIC ZalphaD;

(SEQ ID NO: 54) and

(SEQ ID NO: 55)
This produces the following linker oligonucleotide after annealing:

(SEQ ID NO: 54)

(SEQ ID NO: 55)
-To construct pPIC ZalphaE,

(SEQ ID NO: 56)

(SEQ ID NO: 57)
This produces the following linker oligonucleotide after annealing:

(SEQ ID NO: 56)

(SEQ ID NO: 57)

  These shuttle vectors pPICZalphaD 'and pPICZalphaE' introduced a new cloning site immediately behind the cleavage sites of the respective processing proteases, KEX2 and STE13.

  The E1-H6 coding sequence was isolated from pGENT-ElsH6 (SEQ ID NO: 6, FIG. 1) as an NsI1 / Eco52I fragment. After separation of the digests on a 1% agarose gel, the fragments were purified using a gel extraction kit (Qiagen). The resulting fragment was blunted (using T4 DNA polymerase) and ligated into either pPICZalphaD 'or pPICZalphaE' immediately after the respective processing protease cleavage site.

  The ligation mixture is obtained from E. coli. E. coli TOP10F 'cells were transformed and the plasmid DNA of multiple zeocin resistant colonies was analyzed by restriction enzyme digestion. Positive clones were withheld and designated pPICZalphaD'E1sH6 (ICCG3694; SEQ ID NO: 58, Fig. 32) and pPICZalphaE'ElsH6 (ICCG3475; SEQ ID NO: 59, Fig. 33), respectively.

Construction of pPICZalphaD'E2sH6 and pPICZalphaE'E2sH6 vectors Shuttle vectors pPICZalphaD 'and pPICZalphaE' were constructed as described in Example 11.

  The E2-H6 coding sequence was isolated from pBSK-E2sH6 (SEQ ID NO: 45, FIG. 23) as a SalI / KpnI fragment. After separation of the digests on a 1% agarose gel, the fragments were purified using a gel extraction kit (Qiagen). The resulting fragment was blunted (using T4 DNA polymerase) and ligated into either pPICZalphaD 'or pPICZalphaE' immediately after the respective processing protease cleavage site.

  The ligation mixture is obtained from E. coli. E. coli TOP10F 'cells were transformed and the plasmid DNA of multiple zeocin resistant colonies was analyzed by restriction enzyme digestion. Positive clones were withheld and designated pPICZalphaD'E2sH6 (ICCG3692; SEQ ID NO: 60, Figure 34) and pPICZalphaE'E2sH6 (ICCG3476; SEQ ID NO: 61, Figure 35), respectively.

Transformation of Pichia Pastoris and selection of transformants P. pasta as described in Examples 11 and 12. pastoris shuttle plasmids were purified according to the conditions of the supplier (Invitrogen). Transformed into pastoris cells. E1- and E2-producing strains were reserved for further characterization.

  Well known due to the fact that hyperglycosylation is usually absent (Gellissen, G.2000) and has been used to express Dengue virus E protein as a previous GST fusion (Sugrue, RJ et al., 1997). Yeast strain of P. HCV virus-like particles were expressed in pastoris. Of note, the resulting P.P. pastoris expressed HCV virus-like particles showed glycosylation comparable to that seen in wild-type Saccharomyces strains. More specifically, P.I. HCV virus-like particles produced by pastoris are hyperglycosylated (based on the molecular weight of the expression product detected in a Western blot of proteins isolated from transformed P. pastoris cells).

Culture conditions for Saccharomyces cerevisiae, Hansenula polymorpha and Pichia pastoris Saccharomyces cerevisiae
Cell banking A master cell bank and a working cell bank were prepared from the selected recombinant clones. Cryo-vials were prepared from shake flask culture at mid-logarithmic growth (incubation conditions similar to fermentation seed culture, see below). Glycerol (final concentration 50%) was added as a cryoprotectant.

Fermentation Start a seed culture from a cryopreserved working cell bank and grow it in 500 mL medium (YNB, Difco supplemented with 2% scroll) in a 2 L shake Erlenmeyer flask at 200 rpm, 37 ° C. for 48 hours I let you.

  Fermentation was typically performed in a Biostat C fermentor with a working volume of 15 L (B. Braun Int., Germany, Melsungen, Germany). The fermentation medium contained 1% yeast extract, 2% peptone and 2% sucrose as a carbon source. Polyethylene glycol was used as an antifoaming agent.

Typically temperature, pH and dissolved oxygen were controlled during fermentation. Table 1 summarizes the applicable set points. Dissolved oxygen was cascade controlled by stirring and / or aeration. The pH was controlled by adding NaOH (0.5M) or H ₃ PO ₄ solution (8.5%).

  Fermentation was initiated by the addition of 10% seed culture. During the growth period, sucrose concentration was monitored offline by HPLC analysis (Polysphere Column OAKC Merck).

  During the growth phase, dissolved oxygen was controlled by cascade control (stirring / aeration). After sucrose was completely metabolized, heterogeneous pN production was driven by ethanol produced internally while supplementing with stepwise addition of EtOH to maintain the concentration at about 0.5% (offline HPLC analysis, polysphere OAKC). column). During this induction period, dissolved oxygen was controlled below 5% air saturation by manual adjustment of air flow rate and agitator speed.

  Typically, the fermentation was harvested 48-72 hours after induction by concentration via tangential flow filtration followed by centrifugation of the concentrated cell suspension to obtain a cell pellet. If not analyzed immediately, the cell pellet was stored at -70 ° C.

Hansenula polymorpha
Cell banking A master cell bank and a working cell bank were prepared from the selected recombinant clones. Cryo-vials were prepared from shake flask culture at mid-logarithmic growth (incubation conditions similar to fermentation seed culture, see below). Glycerol (final concentration 50%) was added as a cryoprotectant.

Fermentation Initiated seed culture from a cryopreserved (−70 ° C.) working cell bank and grown in 500 mL medium (YPD, Difco) in 2 L triangular shake flasks at 200 rpm, 37 ° C. for 48 hours. .

  Fermentation was typically performed in a Biostat C fermentor with a 15 L working volume (B. Braun Inc., Melsungen, Germany). The fermentation medium contained 1% yeast extract, 2% peptone and 1% glycerol as a carbon source. Polyethylene glycol was used as an antifoaming agent.

Typically temperature, pH, air and dissolved oxygen were controlled during fermentation. Table 2 summarizes the applicable set points. Dissolved oxygen was cascade controlled by stirring and / or aeration. The pH was controlled by adding NaOH (0.5M) or H ₃ PO ₄ solution (8.5%).

  Fermentation was initiated by the addition of 10% seed culture. During the growth phase, glycerol concentration was monitored off-line (Polysphere Column OAKC merck) and 1% methanol was added 24 hours after complete glycerol consumption to induce heterologous protein expression. Fermentation was harvested 24 hours after induction by concentration via tangential flow filtration followed by centrifugation of the concentrated cell suspension to obtain a cell pellet. If not analyzed immediately, the cell pellet was stored at -70 ° C.

Pichia pastoris
Small scale protein production experiments with recombinant Pichia pastoris were set up in shake flask cultures. Seed cultures were grown overnight in YPD medium (Difco). The pH of the initial medium was corrected to 4.5. The shake flasks were incubated at 37 ° C. and 200-250 rpm on a rotary shaker.

  Small-scale production is typically performed on a 500 mL scale in a 2 L shake flask, on an expression medium containing 1% yeast extract, 2% peptone (both Difco) and 2% glycerol as a carbon source. I started with 10% inoculation. The inoculation conditions were the same as in the seed culture. Induction was initiated by the addition of 1% MeOH approximately 72 hours after inoculation. Cells were harvested 24 hours after induction by centrifugation. If not analyzed immediately, the cell pellet was stored at -70 ° C.

Removal of leader peptide from MFa-E1-H6 and MFa-E2-H6 proteins expressed in selected yeast cells Expression products in Hansenula polymorpha and Saccharomyces cerevisiae glycosylation minus strains of HCV E1 and E2 protein constructs with Cerevisiae a-mating factor (aMF) leader sequences were further analyzed. Both genotypes 1b HCV E1s (aa192-326) and HCV E2s (VIEGR (SEQ ID NO: 69)-aa383-673 extended with the sequence were both C-terminal high tagged (H6, HHHHHH, SEQ ID NO: 63; Since the HCV protein was further expressed in this example as MF-E1-H6 and MF-E2-H6) proteins, the expressed product of yeast cells after solubilization of guanidinilam chloride (GuHCl) was rapidly and efficiently Purification was performed on Ni-IDA (Ni-iminodiacetic acid) Briefly, the cell pellet was resuspended in 50 mM phosphate, 6 M GuHCl, pH 7.4 (9 vol / g cells). 320 mM (4% w / v) sodium sulfite and 65 mM (2% w / v) sodium tetrathionate The protein was sulfonated overnight in the presence of an aqueous solution (RT overnight. The lysate was clarified after a freeze-thaw cycle by centrifugation (10.000 g, 30 min, 4 ° C.) and empigen (Ibright & Wilson (Aibright & Wilson, UK) and imitazole were added to the supernatant to a final concentration of 1% (w / v) and 20 mM, respectively, and the sample was filtered (0.22 μM) and loaded onto a Ni-IDA Sepharose FF column. This was equilibrated with 50 mM phosphate, 6 M GuHCl, 1% empigen (buffer A) supplemented with 20 mM imidazole, and then the column contained 20 mM and 50 mM imidazole, respectively. Was washed to the achieved baseline level of 280 nm using Buffer A. Buffer D, 50 mM The his-tagged product was eluted by applying acid salt, 6M GuHCl, 0.2% (for E1) or 1% (for E2) empigen, 200 mM imidazole. ), Or by SDS-PAGE and Western blot using a specific monoclonal antibody directed against E2 (IGH212).

  The E1 product was analyzed immediately by Edman degradation.

  At this step, SDS-PAGE already revealed a very complex picture of the protein band for HCV E2, so further fractionation by size exclusion chromatography was performed. The Ni-IDA eluate is concentrated by ultrafiltration (MWC 10 kDa, centiplus, Amicon, millipove) and placed on Superdex G200 (10/30 or 16/10; Pharmacia) in PBS, 1% Empigen or PBS, 3% Empigen. I put it in. Elution fractions containing E2 products with Mr between 80 kDa and 45 kDa, ie SDS-PAGE (fractions 17-23 of the elution profile in FIG. 37 based on migration on FIG. 381 were pooled and alkylated. (Incubation with 10 mM DTT for 3 hours at RT followed by incubation with 30 mM iodoacetamide for 3 hours at RT.) Samples for amino-terminal sequencing were treated with EndoH (Roche Biochemicals) or untreated. Glycosylated and deglycosylated E2 products were blotted on PVDF membranes for amino-terminal sequencing, blots stained with glycosylated and deglycosylated E2 amide-black, It is shown in FIG.

  Sequencing of the purified products of both E1 and E2 unfortunately leads to the consideration that removal of the signal sequence from the HCV virus-like particles has only partially occurred (see Table 3). Furthermore, the majority of by-products (products containing degradation products and still leader sequences or parts thereof) are glycosylated. This glycosylation, if partial, is on an uncleaved fragment of the signal sequence that also contains an N-glycosylation site. These sites can be mutated to result in fewer glycosylated byproducts. However, even more problematic is the discovery fact that some products that are alternately cleaved differ by only 1 to 4 amino acids compared to the desired intact envelope protein. As a result, purification of properly processed products is virtually impossible due to the lack of biochemical properties that can be sufficiently discriminated between different expression products. Some of the degradation products are also required for the cleavage of the a-mating factor leader and thus cannot be blocked without disrupting this essential process (eg, the post-arginine cleavage of E1 This may be the result of the cleavage seen after aa196.

  S. pSY1YIG7E1s (SEQ ID NO: 50; FIG. 28). High E1 producing clones derived from transformation of Cerevisiae IYCC155 were obtained from S. cerevisiae with pSY1aMFE1sH6aYIG1E1s (SEQ ID NO: 44; FIG. 22). Comparison with high productivity clones derived from transformation of Cerevisiae IYCC155. Intracellular expression of E1 protein was assessed 2 to 7 days after induction using Western blot with an E1 specific monoclonal antibody (IGH201). As can be seen from FIG. 40, maximum expression was observed after 2 days for both strains, but the expression patterns for both strains are quite different. Expression in the α-mating factor leader results in a very complex band pattern, resulting from the fact that leader processing is not efficient. This leads to multiple expression products with different amino termini and some being modified by 1-5N-glycosylation. However, for E1 expressed in the CL leader, a limited number of completely different bands are visible, indicating that the level of proper CL leader removal is high and from Hansenula where the same CL leader was expressed. Reflects the fact that only this properly processed material can be modified by N-glycosylation (1-5 chains) as observed for E1. (See Example 6).

  A hybridoma cell line that produces a monoclonal antibody directed against E1 (IGH201) is a European cell culture collection agency, Center for Applied Microbiology, Salisbury, Wiltshire SP40 JG, under the conditions of the Budapest Treaty in the UK And deposited on March 12, 1998, with the deposit number ECACC 9803216. A monoclonal antibody directed against E2 (IGH212) is described as antibody 12D11F2 in Example 96 by Maertens et al. In WO96 / 04385.

Expression of the E1 construct in yeast suitable for large scale production and purification To replace the Cerevisiae aMF leader peptide, CHH (Carcinus maenas hyperglycemic hormone leader sequence), Amg1 (amylase leader sequence from S. Occidentalis), Gam1 (glucoamylase leader sequence from S. Occidentalis), Phy5 (fungi Several other leader sequences including a leader sequence from sex phytase), pho1 (leader sequence from Pichia pastoris) and CL (leader of avian lysozyme C, 1,4-beta-N-acetylmuramidase C) , E1-H6 (ie E1 with a C-terminal his-tag). All constructs were expressed in Hansenula polymorpha and each of the resulting cell lysates was subjected to Western blot analysis. This has already led to the conclusion that the extent of removal of the leader or signal sequence or peptide is very low, except for constructs where CL is used as the leader peptide. This was confirmed for the CHH-E1-H6 construct by Edman degradation of the Ni-IDA purified material: multiple different sequences were recovered, but properly cleaved products cannot be detected. None (see Table 4).

  As already mentioned, western blots of cell lysates revealed a pattern of E1-specific protein bands representing a high degree of proper removal of the CL leader peptide. This is surprising because this leader is not derived from yeast. Amino acid sequencing by Edman degradation of material solubilized with GuHCl and purified with Ni-IDA confirmed that 84% of the E1 protein was indeed properly cleaved and the material was essentially free of degradation products. . There is still 16% unprocessed material, but this material is an unglycosylated material that can be easily removed from the mixture, allowing specific enrichment of properly cleaved and glycosylated E1 To do. Such an enrichment method can be affinity chromatography on lectins, other alternatives are also shown in Example 19. Alternatively, the higher hydrophobicity of the unglycosylated material can be used to select and optimize other enrichment procedures. Proper removal of the CL leader peptide from the CL-E1-H6 protein was further confirmed by mass spectrometry, which similarly occupied up to four of the 5N-glycosylation sites of genotype 1bE1s. It has also been confirmed that the sequence NNSS (amino acids 233-236, SEQ ID NO: 73) is thus considered to be a single N-glycosylation site.

Purification and biochemical characterization of HCV E2 protein expressed in Hansenula polymorpha from CL-E2-H6 coding construct CL-E2-VIEGR-H6 expressed in Hansenula polymorpha (hereinafter “CL-E2” in this example) The efficiency of removing the CL leader peptide from the protein was analyzed. Since HCV E2s (aa 383-673) was expressed as a his-tagged protein, efficient high-speed purification of the expressed protein after GuHCl solubilization of the collected cells was performed for Ni-IDA. Briefly, the cell pellet was resuspended in 30 mM phosphate, 6M GuHCl, pH 7.2 (9 mL buffer per gram of cells). The protein was sulfonated overnight at room temperature in the presence of 320 mM (4% w / v) sodium sulfite and 65 mM (2% w / v) sodium tetrathionate. After freeze-thaw cycles with centrifugation (10.000 g, 30 min, 4 ° C. /), the lysate was clarified. Empigen BB (Ibright & Wilson) to final concentrations of 1% (w / v) and 20 mM, respectively. All further chromatographic steps were performed on an AktaFPLC workstation (Pharmacia) Samples were filtered through a 0.22 μm pore size membrane (cellulose acetate) and loaded with a Ni-IDA column (Ni ^2+). This was equilibrated with 1% Empigen BB supplemented with 20 mM imidazole, pH 7.2 (buffer A), 6 M GuHCl, 50 mM phosphate. Buffer A containing 20 mM and 50 mM imidazole, respectively Wash until absorbance at 280 nm reaches baseline level by applying buffer D, 50 mM phosphate, 6 M GHCl, 0.2% Empigen BB (pH 7.2), 200 mM imidazole, The his-tagged product was eluted and the purified material was analyzed by SDS-PAGE and Western blot using a specific monoclonal antibody E2 (IGH212) directed against E2 (FIG. 41). The purified E2-H6 protein was similarly subjected to N-terminal sequencing by Edman degradation, and the protein was further purified by N-glycosidase F (Roche) (0.2 U / μg E2, PBS / 3% Empigen 1 hour incubation at 37 ° C. in BB) or left untreated Glycosylated and deglycosylated E2-H6 proteins were subjected to SDS-PAGE and blotted on PVDF membranes for amino acid sequencing (analysis was performed by Applied Biosystems PROCISE ^™ 492 protein sequencer, At this step, SDS-PAGE revealed some degradation products, so further fractionation by size exclusion chromatography was performed, in which the Ni-IDA eluate was concentrated by ultrafiltration. (MWCO 10 kDa, centriplus, Millipore Amicon (Amicon, Millipore)), PBS, 1% Empigen BB loaded on Superdex G200 (Pharmacia) SDS-PA Based on migration on GE, elution fractions containing primarily intact E2s-related products with Mr between ˜30 kDa and ˜70 kDa were pooled and optionally alkylated (at 37 ° C. for 30 minutes with 5 mM DTT). Followed by incubation with 20 mM iodoacetamide at 37 ° C. for 30 minutes). The possibility of the presence of degradation products after IMAC purification can thus be overcome by further fractionation of intact products using size exclusion chromatography. An unexpectedly excellent result was obtained. Based on N-terminal sequencing, the amount of E2 product from which the CL leader peptide was removed could be estimated. The total amount of protein product is calculated as the pmol of protein based on the intensity of the peak recovered by Edman degradation. Then, for each specific protein (ie, each “detected N-terminus”), the mole percent relative to the sum is estimated. In current experiments, only the proper N-terminus of E2-H6 is detected and contains an N-terminal amino acid that is not contained within the E2 protein, or there are other variants of E2-H6-deficient amino acids of the E2 protein. There wasn't. In conclusion, H.C. The E2-H6 protein expressed as a CL-E2-H6 protein by polymorpha was isolated as 95% or more properly cleaved protein without any further in vitro treatment. This is the H.E. of MF-E2-H6 protein versus E2-H6 protein, estimated to occur in 25% of isolated proteins. This contrasts with the fidelity of leader peptide removal by polymorpha (see Table 3).

Purification and biochemical characterization of HCV E1 protein expressed in Hansenula polymorpha from CL-H6-K-E1 coding construct and in vitro treatment of H6-containing proteins. Efficiency of removal of CL leader peptide from CL-H6-K-E1 protein expressed in Polymorpha and subsequent in vitro to remove H6 (his-tag) -adapter petapeptide and Endo Lys-C processing site The efficiency of processing was analyzed. Since HCV E1s (aa 192-326) was expressed as N-terminal His-K-tagged protein CL-H6-K-E1, high speed and high efficiency purification was performed as described in Example 17. We were able to. The elution profile of IMAC-chromatographic purification of H6-K-E1 (and optionally CL-H6-K-E1) protein is shown in FIG. Specific monoclonal antibody (IGH201) directed against SDS-PAGE and silver staining and E1 of the gel (FIG. 43), elution fractions containing recombinant E1s product (63-69) were pooled (“IMAC Pool)), overnight endoproteinase Lys-C (Roche) treatment (enzyme / substrate ratio 1/50 (w / w), 37 ° C.) to remove the H6-K-fusion tail. Removal of the untreated fusion product is performed by the IMAC chromatography step on the page on the Ni-IDA column, thus collecting the Endo-Lys-C treated protein in the flow-through fraction. In addition, after 10-fold dilution with 10 mM NaH ₂ PO ₄ .3H ₂ O; 1% (v / v) Empigen B, pH 7.2 (Buffer B), endo-blotainase on a Ni-IDA column. Lys-C digested protein samples were applied and then washed with buffer B until the absorbance at 280 nm reached baseline levels. Flow-throughs were collected in the form of different fractions (1-40) and these fractions were screened for the presence of E1s product (FIG. 44). N-terminal H6 (with Mr between ˜15 kDa and ˜30 kDa based on Western blot analysis with migration on SDS-PAGE followed by silver staining or specific monoclonal antibody directed against E1 (IGH201) Fractions (7-28) containing intact E1 with the -K (and possibly residual CL-H6-K) tail removed were pooled and alkylated (5 mM DTT for 30 minutes at 37 ° C). Followed by incubation with 20 mM iodoacetamide at 37 ° C. for 30 minutes).

This material was subjected to N-terminal sequencing (Edman degradation). In addition, protein samples were treated with N-glycosidase F (Roche) (0.2 U / μg E1, PBS / 3% Empigen BB for 1 hour incubation at 37 ° C.) or left untreated. Glycosylated and deglycosylated E1 was then separated by SDS-PAGE and blotted on PDV submembrane for further analysis by Edman degradation (analysis was performed on the PROCISE ^™ 492 protein sequencer of the Applied Pio system ). Based on N-terminal sequencing, the amount of properly processed E1 product could be estimated (processing involves proper cleavage of the H6-K-sequence). The total amount of protein product is calculated as the pmol of protein based on the intensity of the peak recovered by Edman degradation. Then, for each specific protein (ie, each “detected N-terminus”, the mole% relative to the sum is estimated. In the current experiment, only the proper N-terminus of E1 was detected and H6-K-E1 The N-terminus of other processing variants is not detected, based on the in vitro processing of Endo Lys-C of H6-K-E1E1 (and possibly residual CL-H6-K-E1) protein against E1 protein, It was estimated to occur with a fidelity greater than 95%.

Specific removal of the hypoglycosylated form of HCV E1 by heparin To find a specific purification step for HCV virus-like particles from yeast cells, binding to heparin was evaluated. Heparin has been shown to bind to multiple viruses, and as a result, binding to HCV virus-like particles has already been suggested (Garson, JA et al., 1999). To analyze this potential binding, heparin was biotinylated and Sulfonated HCV E1, H. from Polymopha. Of alkylated HCV E1 from Polymorpha (both produced as described in Example 16) and alkylated HCV E1 from cultures of mammalian cells transfected with a vaccinia expression vector Interaction with HCV E1 was analyzed in microtiter plates coated with either. Surprisingly, strong bonds are Only observed with sulfonated HCV E1 from Polymorpha, while there was no binding with HCV E1 from mammalian cell culture. Using Western blots we were able to show that this binding is specific for the lower molecular weight band of the HCV E1s protein mixture corresponding to the mature HCV E1 that was hypoglycosylated (FIG. 45). . FIG. 45 also demonstrates that this sulfonation is not essential for heparin binding, since the binding is still observed for low molecular weight E1 even when sulfonation is removed (lane 4). Alternatively, alkylation substantially reduces this linkage, which can be caused by the specific alkylating agent used in this example (iodo-acetamide). Surprisingly, it is specifically possible to enrich HCV E1 preparations to obtain preparations with HCV E1 proteins with a high degree of glycosylation (ie occupying more glycosylation sites) As such, this finding further demonstrated the industrial applicability of the CL-HCV-envelope expression cassette for yeast.

Formation and analysis of virus-like particles (VLP) The conversion of HCV E1 and E2 proteins expressed in Polymorpha (Examples 16-18) to VLPs is essentially as described by Depra et al. In WO 99/67285 and by Bosman et al. In WO 01/30815, As described by. Briefly, transformed H. cerevisiae cells expressing HCV virus-like particles. After culture of Polymorpha cells, the cells were harvested and lysed and sulfonated in GuHCl as described in Example 17. The His-tagged protein was then purified by IMAC and concentrated by ultrafiltration as described in Example 17.

VLP formation of HCV virus-like particles with sulfonated Cys-thiol groups Concentrated HCV virus-like particles sulfonated during the isolation procedure were not subjected to reduction treatment with PBS, 1% (v / v) empigen. Loaded onto an equilibrated size exclusion chromatography column (Superdex G200, pharmacia). The eluted fractions were analyzed by SDS-PAGE and Western blotting. Fractions with a relative Mr˜29−˜15 kD (based on SDS-PAGE migration) are pooled, concentrated, loaded onto Superdex G200, equilibrated with PBS, 3% (w / v) betaine, and virus-like Particles (VLP) were formed. Fractions were pooled, concentrated and desalted against PBS, 0.5% (w / v) betaine.

VLP formation of HCV virus-like particles with irreversibly modified Cys-thiol groups Reduced treatment of concentrated HCV virus-like particles sulfonated during the isolation procedure (incubation in the presence of 5 mM DTT in PBS) In contrast, the sulfonated Cys-thiol group was converted to a free Cys-thiol group. Irreversible by (i) 30 min incubation in the presence of 20 mM iodoacetamide or (ii) 30 min incubation in the presence of 5 mM N-ethylmaleimide (NEM) and 15 mM biotin-N-ethylmaleimide Cys-thiol modification was performed. Subsequently, a size exclusion chromatography column equilibrated with PBS, 1% (v / v) empigen, or NEM and biotin-NEM for blocking with iodoacetamide, PBS, 0.2% CHAPS ( The protein was loaded onto Superdex G200 (Pharmacia). The eluted fractions were analyzed by SDS-PAGE and Western blotting. Fractions with a relative Mr of ˜29--15 kD (based on SDS-PAGE migration) are pooled and concentrated to form virus-like particles (VLP) with PBS, 3% (w / v) betaine. Loaded onto an equilibrated Superdex G200. Fractions are pooled and concentrated to PBS, 0.5% (w / v) betaine for iodoacetamide blockade, or 0.05% for block with NEM and bioten-NEM. Desalted with CHAPS.

VLP formation of HCV virus-like particles with reversibly modified Cys-thiol groups Reduced treatment of concentrated HCV virus-like particles sulfonated during the isolation procedure (incubation in the presence of 5 mM DTT in PBS) The sulfonated Cys-thiol group was converted to a free Cys-thiol group. Reversible Cys-thiol modification was performed by incubation for 30 minutes in the presence of dithiodipyridine (DTDP), dithiocarbamate (DTC) or cysteine. The protein was then loaded onto a size exclusion chromatography column (Superdex G200, Pharmacia) equilibrated with PBS, 1% (v / v) empigen. The eluted fractions were analyzed by SDS-PAGE and Western blotting. Fractions with a relative Mr of ˜29--15 kD (based on SDS-PAGE migration) are pooled and concentrated to form virus-like particles (VLP) with PBS, 3% (w / v) betaine. Loaded onto an equilibrated Superdex G200. Fractions were pooled, concentrated and desalted against PBS, 0.5% (w / v) betaine.

  H. Size exclusion chromatography elution profiles in PBS, 3% (w / v) betaine to obtain a VLP of E2-H6 expressed in Polymorpha are shown in FIG. 46 (sulfonated) and FIG. 47 (alkylated with iodoacetamide). Shown in

  H. Size elution chromatography elution profiles in PBS, 3% (w / v) betaine to obtain E1 VLP expressed in Polymorpha are shown in FIG. 48 (sulfonated) and FIG. 48 (alkylated with iodoacetamide). It is shown in The resulting VLPs were analyzed by SDS-PAGE and Western blotting as shown in FIG.

H. Size analysis of VLPs formed by HCV virus-like particles expressed in Polymorpha VLP particle size was determined by dynamic light scattering. For light scattering experiments, photon correlation spectroscopy (PCS, software controlled particle size analyzer (Zetasizer 1000 HS, Malvern Instruments Ltd., Malvern, Worcester UK) Photon correlation spectroscopy or dynamic light scattering (DLS) is an optical method that measures Brownian motion and correlates it to particle size. Guided through the entire macromolecule or particle moving in accordance with Brownian motion, a portion of the laser light is scattered by the particle, and this scattered light is measured by a photomultiplier tube. Is fed into the correlator so that appropriate data analysis is performed. An autocorrelation function is generated that is passed to a computer that was used, and the laser used was a 10 mw monochromatic coherent He-Ne laser with a fixed wavelength of 633 nm, 3 to 6 consecutive samples for each sample. Measurements were made and the results of these experiments are summarized in Table 5.

  H. The observation that sulfonated HCV E1 from polymorpha still forms particles with one size within the same range as alkylated HCV E1 from Hansenula is surprising. Since the net increase as a result of sulfonation (up to 8 Cys-thiol groups can be modified on HCV E1) net increase should induce ionic repulsion between subunits, such an effect is , Was not expected. Other reversible cysteine-modifying substances tested also allowed particle formation, but the HCV E1 produced in this way is less stable than the sulfonated material and is based on the disulfide-based HCV E1 It was proved to result in agglomeration. In order to use these other reversible blocking agents, further optimization of the conditions is required.

Antigenic equivalence of HCV E1-H6 produced by Hansenula and HCV E1 produced by mammalian cells infected with vaccinia The reactivity of serum from HCV chronic carriers with Hansenula-produced HCV E1-H6 was Compared to the reactivity of HCV E1 produced by mammalian cells infected with HCV recombinant vaccinia virus as described by (Depra) et al. Both HCV-E1 preparations tested consisted of VLPs in which the HCV E1 protein was alkylated with NEM and hyotin-NEM. The reactivity of both HCV E1 VLP preparations with serum from HCV chronic carriers was determined by ELISA. The results are summarized in Table 6. As can be derived from Table 6, HCV E1 expressed in HCV recombinant vaccinia virus infected mammalian cells, There was no difference in reactivity between HCV E1 expressed in polymorpha.

Immunogenic equivalence of HCV E1-H6 produced by Hansenula and HCV E1 produced by vaccinia-infected mammalian cells As compared to the immunogenicity of HCV E1 produced by HCV recombinant vaccinia infected mammalian cells. Both HCV-E1 preparations tested consisted of VLPs in which the HCV E1 protein was alkylated with iodoacetamide. Both VLP preparations were formulated with alum and injected into Balb / c mice (5 μg E1 in 125 μl each containing 0.13% Alhydrogel, Superfos, Denmark, each 3 weeks apart) 3 intramuscular / subcutaneous injections). Mice were bled 10 days after the third immunization.

  The results of this experiment are shown in FIG. For the upper part of FIG. 51, the antibodies generated after immunization with E1 VLPs produced in mammalian cells were determined. An ELISA method in which either E1 produced in mammalian cells (“M”) or Hansenula produced E1 (“H”) is coated directly onto an ELISA solid support and then blocked with casein (Example 21). The antibody titer was determined by reference). For the lower part of FIG. 51, the antibodies generated after immunization with Hansenula-produced E VLPs were determined. An ELISA method in which either E1 produced in mammalian cells (“M”) or Hansenula produced E1 (“H”) is coated directly onto an ELISA solid support and then blocked with casein (Example 21). The antibody titer was determined by reference).

  The antibody titer determined was the endpoint titer. The endpoint titer is determined as the dilution of serum that results in an OD (determined by ELISA) equal to twice the mean of the assay background.

  FIG. 51 shows that no significant difference was observed between the immunogenic properties of both E1 compositions and the determined antibody titer was used in the ELISA to perform the endpoint titer. It shows that it is unrelated to the antigen.

  HCV E1 from yeast induced a protective response similar to that obtained at the time of vaccination with alkylated HCV E1 derived from mammalian cell culture at the time of vaccination. The latter response could prevent the chronic progression of HCV after acute infection.

Antigenicity and immunogenicity of sulfonated Hansenula-produced HCV E1-H6 The reactivity of HCV chronic carrier sera with Hansenula-produced HCV E1-H6 sera as described by Depla et al. Comparison was made with the reactivity of HCV E1 produced by HCV recombinant vaccinia virus infected mammalian cells. Both tested HCV-E1 preparations consisted of VLPs in which the HCV E1 protein produced by Hansenula was sulfonated and the HCV E1 produced by mammalian cells was alkylated. The results are shown in Table 7. The overall (average) reactivity was the same, but some major differences were noted for individual sera. This implies that the sulfonated material presents at least some of its epitopes differently than the alkylated HCV E1.

  The immunogenicity of HCV E1-H6 produced by sulfonated Hansenula was compared to the immunogenicity of HCV E1-H6 produced by alkylated Hansenula. Both HCV-E1 preparations tested consisted of VLPs. Both VLP preparations were formulated with alum and injected into Balb / c mice. (Three intramuscular / subcutaneous injections consisting of 5 μg E1 in 125 μl each containing 0.13% Alhydrogel, Superfos, Denmark each 3 weeks apart). Mice were bled 10 days after the third immunization.

  Antibody titers were determined as described in Example 22. Surprisingly, immunization with sulfonated materials resulted in higher antibody titers, regardless of the antigen used in the ELISA to assess these titers (FIG. 51). : Upper plate: Titration of antibody generated against alkylated E1; Lower plate: Titration of antibody generated against sulfonated E1; Alkylated coated on ELISA plate “S”: sulfonated E1 coated on ELISA plate). In this experiment, however, individual titers depend on the antigen used for the analysis confirming the observations obtained on sera from HCV patients. Thus, HCV E1, in which the cysteine thiol group is reversibly modified, may be more immunogenic and thus have an increased efficacy as a vaccine protecting against HCV (chronic infection). In addition, the induction of a response to a neoepitope induced by irreversible blockade is unlikely to occur.

HCV E1-H6 produced in Hansenula and HCV E1 produced by mammalian cells infected with vaccinia with sera from vaccinated chimpanzees HCV recombinant vaccinia virus infected mammalian cells The reactivity of E1 produced by E. coli and E1-H6 produced by Hansenula (both alkylated) with sera and monoclonal antibodies from vaccinated chimpanzees were compared. Furthermore, the E1 protein was coated directly on the plate in the ELISA, and then the plate was saturated with casein. The endpoint titer of the antibody that binds the E1 protein coated to the ELISA plate was determined for specific mouse monoclonal antibodies and chimpanzee sera obtained from animals immunized with E1, all produced by mammalian cells. End point titer determination was performed as described in Example 22. The mouse monoclonal antibodies used were IGH201 (see Example 15), IGH198 (IGH198 = Maertens et al. In WO96 / 04385, 23C12), IGH203 (IGH203 = Maertens et al., WO96 / 04385 in 15G6) and IGH202 (IGH202 = Maertens et al., 3F3 in WO99 / 50301).

  As can be derived from FIG. 53, the reactivity of seven different chimpanzees is identical when tested with E1 protein produced by either Hansenula or mammalian cells. Similarly, the reactivity of monoclonal antibodies against HCV E1 is approximately equal. Two of the chimpanzees (Yoran and Marti) were involved in the prophylactic vaccine study and were able to clear the acute infection at the time of challenge, whereas the control animals failed to clear the infection. The other five chimpanzees (Ton, Phil, Marcel, Peggy, Fema) are involved in therapeutic vaccination studies, and at the time of HCV E1 immunization, the histological activity indicators on liver biopsies and / or It showed reduced liver damage as measured by ALT in the serum.

  The results obtained in this experiment expressed ECVE2 protein in both Saccharomyces cerevisiae and Kluyveromyces lactis. This is clearly different from the findings of Mustilli and collaborators (Mustilli AC et al., 1999). However, E2 produced by purified yeast was chimpanzee immunized with HCV E2 produced by mammalian cells, although the reactivity with monoclonal antibodies was higher than for HCV E2 produced by yeast. Was different from HCV E2 produced by mammalian (CHO) cells in that lower reactivity was observed with sera from.

Glucose profiling of HCV E1 by phosphor-assisted carbohydrate electrophoresis (FACE) Glycosylation profile produced by mammalian cells infected with HCV recombinant vaccinia virus as described by Depra et al. In WO 99/67285. HCV E1 and Hansenula-produced HCV E1 were compared. This was done using phosphor-assisted carbohydrate electrophoresis (FACE). Furthermore, oligosaccharides were released from mammalian cells or E1s produced by Hansenula by peptide-N-glycidase (protein Gase F) and labeled with ANTS (alkylating E1 protein with iodoacetamide prior to PNGase F digestion). did). The ANTS-labeled oligosaccharides were separated by PAGE on 21% polyacrylamide at 15 mA current for 2-3 hours at 4 ° C. It was concluded from FIG. 54 that E1 produced by mammalian cells and oligosaccharides on E1-H6 produced by Hansenula migrate like oligomaltose with a degree of polymerization of 7-11 monosaccharides. This means that the Hansenula expression system surprisingly has E1 proteins that are not hyperglycosylated and have a sugar chain similar in length to the sugar chains added to the E1 protein produced in mammalian cells. It shows that it leads.

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  The present invention provides a recombinant nucleic acid encoding an HCV envelope protein and an avian lysozyme leader peptide, a method for producing an HCV envelope protein, and the like, which are useful in the pharmaceutical field for the prevention or treatment of hepatitis C.

FIG. 7 is a schematic map of the vector pGEMT-E1sH6RB having the sequence as defined in SEQ ID NO: 6. FIG. 10 is a schematic map of vector pCHH-Hir having a sequence as defined in SEQ ID NO: 9. FIG. 6 is a schematic map of vector pFPMT121 having a sequence as defined in SEQ ID NO: 12. Figure 2 is a schematic map of the vector pFPMT-CHH-E1-H6 having the sequence as defined in SEQ ID NO: 13. FIG. 6 is a schematic map of vector pFPMT-MFa-E1-H6 having a sequence as defined in SEQ ID NO: 16. FIG. 6 is a schematic map of vector pUC18-FMD-MFa-E1-H6 having the sequence as defined in SEQ ID NO: 17. FIG. 6 is a schematic map of vector pUC18-FMD-CL-E1-H6 having a sequence as defined in SEQ ID NO: 20. FIG. 2 is a schematic map of vector pFPMT-CL-E1-H6 having a sequence as defined in SEQ ID NO: 21. Figure 2 is a schematic map of vector pSP72E2H6 having the sequence as defined in SEQ ID NO: 22. FIG. 4 is a schematic map of vector pMPT121 having the sequence as defined in SEQ ID NO: 23. FIG. 6 is a schematic map of vector pFPMT-MFa-E2-H6 having the sequence as defined in SEQ ID NO: 24. FIG. 6 is a schematic map of the vector pMPT-MFa-E2-H6 having the sequence as defined in SEQ ID NO: 25. FIG. 29 is a schematic map of vector pMF30 having a sequence as defined in SEQ ID NO: 28. FIG. 3 is a schematic map of the vector pFPMT-CL-E2-H6 having the sequence as defined in SEQ ID NO: 32. FIG. 40 is a schematic map of vector pUC18-FMD-CL-E1 having the sequence as defined in SEQ ID NO: 35. FIG. 7 is a schematic map of vector pFPMT-CL-E1 having a sequence as defined in SEQ ID NO: 36. 40 is a schematic map of the vector pUC18-FMD-CL-H6-E1-K-H6 having the sequence as defined in SEQ ID NO: 39. FIG. 4 is a schematic map of the vector pFPMT-CL-H6-K-E1 having the sequence as defined in SEQ ID NO: 40. FIG. 4 is a schematic map of vector pYIG5 having the sequence as defined in SEQ ID NO: 41. FIG. 4 is a schematic map of vector pYIG5E1H6 having the sequence as defined in SEQ ID NO: 42. FIG. 4 is a schematic map of vector pSY1 having a sequence as defined in SEQ ID NO: 43. FIG. 4 is a schematic map of the vector pSY1aMFE1sH6a having the sequence as defined in SEQ ID NO: 44. FIG. 4 is a schematic map of vector pBSK-E2sH6 having the sequence as defined in SEQ ID NO: 45. FIG. 2 is a schematic map of vector pYIG5HCCL-22aH6 having the sequence as defined in SEQ ID NO: 46. FIG. 46 is a schematic map of vector pYYIGSE2H6 having the sequence as defined in SEQ ID NO: 47. FIG. 4 is a schematic map of vector pYIG7 having the sequence as defined in SEQ ID NO: 48. FIG. 5 is a schematic map of vector pYIG7E1 having the sequence as defined in SEQ ID NO: 49. FIG. 6 is a schematic map of vector pSY1YIG7E1s having a sequence as defined in SEQ ID NO: 50. FIG. 6 is a schematic map of the vector pPICZalphaA having the sequence as defined in SEQ ID NO: 51. FIG. 6 is a schematic map of the vector pPICZalphaD ′ having the sequence as defined in SEQ ID NO: 52. FIG. 4 is a schematic map of the vector pPICZalphaE ′ having the sequence as defined in SEQ ID NO: 53. FIG. 6 is a schematic map of the vector pPICZalphaD′E1sH6 having the sequence as defined in SEQ ID NO: 58. FIG. 6 is a schematic map of the vector pPICZalphaE′E1sH6 having the sequence as defined in SEQ ID NO: 59. FIG. 6 is a schematic map of the vector pPICZalphaD′E2sH6 having the sequence as defined in SEQ ID NO: 60. FIG. 6 is a schematic map of the vector pPICZalphaE′E2sH6 having the sequence as defined in SEQ ID NO: 61. FIG. 6 is a schematic map of the vector pUC18MFa having the sequence as defined in SEQ ID NO: 62. Size exclusion chromatography elution profile of purified E2-H6 protein by IMAC expressed from Hansenula polymorpha expressing MFa-E2-H6 (see Example 15). The X axis represents the elution volume (in mL). The vertical line through the elution profile represents the collected fraction. “P1” = pooled fractions 4-9, “P2” = pooled fractions 30-35 and “P3” = pooled fractions 37-44. The Y axis represents the absorbance given in mAu (milli absorbance units). The X axis represents the elution volume in mL. Different pools and fractions collected after size exclusion chromatography (see Figure 37) were analyzed by non-reducing SDS-PAGE followed by silver staining of polyacrylamide gels. The analyzed pools (“P1”, “P2” and “P3”) and fractions (16-26) are shown at the top of the silver stained gel picture. On the left (lane “M”), the size of the molecular weight marker is shown. Fractions 17-23 of the size exclusion chromatography step as shown in FIG. 37 were pooled and alkylated. The protein material was then subjected to Endo H treatment for deglycosylation. Untreated material and Endo H-treated material were separated on an SDS-PAGE gel and blotted to a PVDF membrane. The blot was stained with amide black. Lane 1: alkylated E2-H6 before Endo H-treatment Lane 2: alkylated E2-H6 after Endo H-treatment Western blot analysis of E1 cell lysates expressed in Saccharomyces cerevisiae. Western blots were developed using E1-specific monoclonal antibody IGH201. Lanes 1-4: 2 days, 3 days, respectively, in a Saccharomyces clone transformed with pSY1YIG7E1s (SEQ ID NO: 50, FIG. 28) containing a nucleotide sequence encoding a chicken lysozyme leader peptide linked to E1-H6. Expression product after 5 or 7 days of expression. Lanes 5-7: 2 days, 3 days, respectively, in Saccharomyces clones transformed with pSY1aMFE1sH6aYIGI (SEQ ID NO: 44, FIG. 22) containing the nucleotide sequence encoding the a-mating factor leader peptide bound to E1-H6 Expression product after day or 5 days of expression. Lane 8: Molecular weight marker with the indicated size Lane 9: Purified E1 produced by mammalian cells infected with HCV-recombinant vaccinia virus. H. E2-H6 protein analysis purified by immobilized metal ion affinity chromatography (IMAC) expressed by polymorpha and thus processed from CL-E2-H6 to E2-H6 (see Example 17). Proteins in different wash fractions (lanes 2-4) and elution fractions (lanes 5-7) are directed against reducing SDS-PAGE followed by silver staining of the gel (A, top) or E2. Were analyzed by Western blotting using the specific monoclonal antibody (B, bottom photo). The size of the molecular weight marker is marked on the left. H. Elution profile of the first IMAC chromatography step on a Ni-IDA column for purification of the sulfonated H6-K-E1 protein produced by polymorpha (chelated Sepharose FF loaded with Ni ²⁺ , Pharmacia) (See Example 18). The column was equilibrated with Buffer A supplemented with 20 mM imidazole (50 mM phosphate, 6 M GuHCl, 1% Empigen BB (v / v), pH 7.2). After sample application, the column was washed sequentially with Buffer A containing 20 mM and 50 mM imidazole, respectively (as shown on the chromatogram). Further washing and elution steps of His-tagged products were performed (as indicated on the chromatogram) Buffer B supplemented with 50 mM imidazole and 200 mM imidazole, respectively (PBS 1% epigen BB, pH 7. 2) was performed sequentially. The following fractions were pooled: Wash Pool 1 (Fraction 8-11, wash with 50 mM imidazole). The eluted material was collected as separate fractions 63-72 or an elution pool (fractions 63-69) was prepared. The Y axis represents the absorbance given in mAU (milli absorbance units). The X axis shows the elution volume in mL. H. Analysis of IMAC purified H6-K-E1 protein expressed by polymorpha and processed from CL-H6-K-E1 to H6-K-E1 (see FIG. 42). Proteins in wash pool 1 (lane 12) and elution fractions 63-72 (lanes 2-11) were analyzed by reducing SDS-PAGE followed by silver staining of the gel (A, top photo). By Western blotting using a specific monoclonal antibody directed against E1 (IGH201), in the sample before IMAC (lane 2), in the flow-through pool (lane 4), in wash pool 1 (lane 5), And the protein present in the elution pool (lane 6) was analyzed (B, bottom photo; no sample was loaded in lane 3). The size of the molecular weight marker (lane M) is shown on the left. Elution profile of a second IMAC chromatography step on a Ni-IDA column for purification of E1 resulting in in vitro treatment of H6-K-E1 with Endo Lys-C (purification: see FIG. 42) (Chelated Sepharose FF charged with Ni ²⁺ , Pharmacia). The flow-through was collected in the form of different fractions (1-40) and screened for the presence of E1-product. Fractions (7-28) containing intact E1 processed from H6-K-E1 were pooled. It represents the absorbance given in mAU (milli absorbance units). The X axis shows the elution volume in mL. Western blot analysis showing specifically E1s protein bands that react with biotinylated heparin (see also Example 19). Purified from a mammalian culture infected with HCV recombinant vaccinia virus or The E1s preparation expressed by polymorpha was analyzed. The plate to the right of the vertical line shows a Western blot developed with biotinylated E1-specific monoclonal IGH200. The plate on the left from the vertical line shows a Western blot developed with biotinylated heparin. These results led to the conclusion that E1s, which have undergone relatively low glycosylation, have a high affinity for heparin. Lane M; molecular weight marker (molecular weight is shown on the left). Lane 1; E1s from mammalian cells alkylated during isolation. Lane 2; E1s-H6 expressed by polymorpha and sulfonated during isolation. Lane 3; E1s-H6 expressed by polymorpha and alkylated during isolation. Lane 4; same material as charged in lane 2, but treated with cythiothreitol to convert the sulfonated Cys-thiol group to Cys-thiol. The sulfonated form of purified H.sub.2 subjected to a run in PBS, 3% betaine to form virus-like particles by exchange of Empigen BB for betaine. Size exclusion chromatographic (SEC) profile of E2-E6 expressed in polymorpha. Pooled fractions containing VLPs used for further studies are represented by “← →”. The Y axis shows the absorbance given in mAU (milli absorbance units). The X axis represents the elution volume expressed in mL. See also Example 20. The exchange of Empigen BB for betaine results in an alkylated form of purified H.sub.2 subjected to a run in PBS, 3% betaine to form virus-like particles. Profile by size exclusion chromatography (SEC) of E2-E6 expressed in polymorpha. Pooled fractions containing VLPs are represented by “← →”. The Y axis shows the absorbance given in mAU (milli absorbance units). The X axis represents the elution volume expressed in mL. See also Example 20. The sulfonated form of purified H.sub.2 subjected to a run in PBS, 3% betaine to form virus-like particles by exchange of Empigen BB for betaine. Profile by size exclusion chromatography (SEC) of E1 expressed in polymorpha. Pooled fractions containing VLPs are represented by “← →”. The Y axis shows the absorbance given in mAU (milli absorbance units). The X axis represents the elution volume expressed in mL. See also Example 20. The exchange of Empigen BB for betaine results in an alkylated form of purified H.sub.2 subjected to a run in PBS, 3% betaine to form virus-like particles. Profile by size exclusion chromatography (SEC) of E1 expressed in polymorpha. Pooled fractions containing VLPs are represented by “← →”. The Y axis shows the absorbance given in mAU (milli absorbance units). The X axis represents the elution volume expressed in mL. See also Example 20. SDS-PAGE (under reducing conditions) and Western blot analysis of VLPs as isolated after size exclusion chromatography (SEC) as described in FIGS. 48 and 49. Left panel: Silver-stained SDS-PAGE gel. Right panel: Western blot using a specific monoclonal antibody (IGH201) directed against E1. Lane 1: molecular weight marker (molecular weight is shown on the left); Lane 2: pool of VLPs containing sulfonated E1 (see FIG. 48); Lane 3: pool of VLPs containing alkylated E1 (FIG. 49). reference). See also Example 20. Endpoints of antibodies present in serum after vaccination of mice with E1 produced in mammalian cells or (upper plate) or after vaccination of mice with E1 produced in Hansenula (lower plate) To determine the titer, an ELISA solid support was coated with E1 ("M") produced in mammalian cells or E1 ("H") produced in Hansenula. Horizontal bars represent mean antibody titers. The end point titer (dilution factor) is represented on the Y-axis. See also Example 22. Hansenula-produced E1 is alkylated (“A” or (“S”)) after vaccination of mice with alkylated Hansenula-produced E1 (on the plate) or with sulfonated Hansenula-produced E1 Coated on ELISA solid support to determine the end point titer of antibody present in serum after vaccination (bottom plate) Horizontal bar represents mean antibody titer. ) Is represented on the Y-axis, see also Example 23. HCV E1 and H. coli produced by mammalian cells infected with HCV recombinant vaccinia virus. HCV E1 produced by polymorpha was coated directly on the ELISA plate. Mouse monoclonal antibody raised against E1 produced by mammalian cells (bottom) and endpoint titer of antibody in the serum of chimpanzee vaccinated with E1 produced by mammalian cells (top) It was determined. Chimpanzees Yoran and Marti were vaccinated prophylactically. Chimpanzees Ton, Phil, Marcel, Peggy and Fema were therapeutically vaccinated. Black bar: E1 coated ELISA plate produced by mammalian cells. White bar: E1 coated ELISA plate produced by Hansenula. The end point titer (dilution factor) is shown on the Y-axis. See also Example 24. Fluorophore-assisted carbohydrate gel electrorheology of oligosaccharides released from E1 produced by mammalian cells infected with recombinant vaccine virus and E1-H6 protein produced by Hansenula. Lane 1: Glucose ladder standard with the number of monosaccharides (3-10, indicated by G3-G10) shown on the left. Lane 2: 25 μg N-linked oligosaccharide released from (alkylated) E1 produced by mammalian cells. Lane 3: 25 μg N-linked oligosaccharide released from (alkylated) E1-H6 produced by Hansenula. Lane 4: 100 pmoles of maltotetraose. See also FIG.

SEQ ID NO: 1; avian lysozyme signal peptide; Xaa is Arg, Lys or Val; Xaa is Ser, Ala, Val, Arg or Met; Xaa is Leu or Phe; Xaa is Leu or Ala; Xaa is Ile, Thr, Phe or Val; Xaa is Leu, Phe or Ala; Xaa is Val, Ile, Ala, Leu or Cys; Xaa is Leu, Phe, Ala or Ile; Xaa is Cys, Phe, Ser or Leu; Xaa is Phe, Leu, Ser or Pro; Xaa is Leu, Ala or Met; Xaa is Pro, Ala or Ile; Xaa is Leu or Ala; Xaa is Ala, Val, Ser or Met; Xaa is Ala, Lys or Ser; Xaa is Leu, Pro, Gln or Ile
SEQ ID NO: 6; vector pGEMTE1sH6
SEQ ID NO: 7; synthetic probe or primer
SEQ ID NO: 8; synthetic probe or primer
SEQ ID NO: 9; vector pCHH-Hir
SEQ ID NO: 10; synthetic probe or primer
SEQ ID NO: 11; synthetic probe or primer
SEQ ID NO: 12; vector pFPMT121
SEQ ID NO: 13; vector pFPMT-CHH-E1H6
SEQ ID NO: 14; synthetic probe or primer
SEQ ID NO: 15; synthetic probe or primer
SEQ ID NO: 16; vector pFPMT-Mfalfa-E1-H6
SEQ ID NO: 17; vector pUC18-FMD-MFalfa-E1-H6; N is any nucleotide; N is any nucleotide
SEQ ID NO: 18; synthetic probe or primer
SEQ ID NO: 19; synthetic probe or primer
SEQ ID NO: 20; vector pUC18-FMD-CL-E1-H6; N is any nucleotide; N is any nucleotide
SEQ ID NO: 21; vector pFPMT-CL-E1-H6
SEQ ID NO: 22; vector pSP72E2H6
SEQ ID NO: 23; vector pMPT121; N is any nucleotide
SEQ ID NO: 24; vector pFMPT-MFalfa-E2-H6
SEQ ID NO: 25; vector pMPT-Mfalfa-E2-H6; N is any nucleotide
SEQ ID NO: 26; synthetic probe or primer
SEQ ID NO: 27; synthetic probe or primer
SEQ ID NO: 28; vector pMF30
SEQ ID NO: 29; synthetic probe or primer
SEQ ID NO: 30; synthetic probe or primer
SEQ ID NO: 31; synthetic probe or primer
SEQ ID NO: 32; vector pFMPT-CL-E2-H6
SEQ ID NO: 33; synthetic probe or primer
SEQ ID NO: 34; synthetic probe or primer
SEQ ID NO: 35; vector pUC18-FMD-CL-E1; N is any nucleotide; N is any nucleotide
SEQ ID NO: 36; vector pFPMT-CL-E1
SEQ ID NO: 37; synthetic probe or primer
SEQ ID NO: 38; synthetic probe or primer
SEQ ID NO: 39; vector pUC18-FMD-CL-E1-H-K6; N is any nucleotide; N is any nucleotide
SEQ ID NO: 40; vector pFPMT-CL-H6-K-E1; N is any nucleotide: N is any nucleotide
SEQ ID NO: 41; vector pYIG5
SEQ ID NO: 42; vector pYIG5E1H6
SEQ ID NO: 43; vector pSY1
SEQ ID NO: 44; vector pSY1AMFE1sH6a
SEQ ID NO: 45; vector pBKS-E2sH6
SEQ ID NO: 46; vector pYIG5HCCL-22aH6
SEQ ID NO: 47; vector pYYIGSE2H6
SEQ ID NO: 48; vector pYIG7
SEQ ID NO: 49; vector pYIG7E1
SEQ ID NO: 50; vector pSY1YIG7E1s
SEQ ID NO: 51; vector pPICZalphaA
SEQ ID NO: 52; vector pPICZalphaD '
SEQ ID NO: 53; vector pPICZalphaE '
SEQ ID NO: 54; synthetic probe or primer
SEQ ID NO: 55; synthetic probe or primer
SEQ ID NO: 56; synthetic probe or primer
SEQ ID NO: 57; synthetic probe or primer
SEQ ID NO: 58; vector pPICZalphaD 'E1sH6
SEQ ID NO: 59; vector pPICZalphaE 'E1sH6
SEQ ID NO: 60; vector pPICZalphaD 'E2sH6
SEQ ID NO: 61; vector pPICZalphaE 'E2sH6
SEQ ID NO: 62; vector pUC18MFa
SEQ ID NO: 63; adaptor peptide
SEQ ID NO: 64; adaptor peptide
SEQ ID NO: 65; adaptor peptide
SEQ ID NO: 66; processing site; X is any amino acid
SEQ ID NO: 67; processing site; X is any amino acid
SEQ ID NO: 68; processing site; X is any amino acid
SEQ ID NO: 69; adaptor peptide
SEQ ID NO: 70; adaptor peptide
SEQ ID NO: 71; adaptor peptide
SEQ ID NO: 72; adaptor peptide
SEQ ID NO: 73; HCV E1
SEQ ID NO: 74; FLAG epitope
SEQ ID NO: 75; Protein C epitope
SEQ ID NO: 76; VSV epitope
SEQ ID NO: 77; streptag
SEQ ID NO: 78; Tag100 epitope
SEQ ID NO: 79; c-myc epitope
SEQ ID NO: 80; HA epitope
SEQ ID NO: 81; HA epitope
SEQ ID NO: 82; HA epitope
SEQ ID NO: 83; thrombin cleavage site
SEQ ID NO: 84; collagenase recognition site; Xaa is any amino acid but most frequently a neutral amino acid

Claims (36)

  A recombinant nucleic acid comprising a nucleotide sequence encoding a protein comprising an avian lysozyme leader peptide or a functional equivalent thereof bound to an HCV envelope protein or a portion thereof. The protein is:
CL-[(A1) _a- (PS1) _b- (A2) _c ] -HCVENV-[(A3) _d- (PS2) _e- (A4) _f ]
It is characterized by the structure
In the formula;
CL is an avian lysozyme leader peptide or a functional equivalent thereof;
A1, A2, A3 and A4 are adapter peptides that can be different or the same;
PS1 and PS2 are processing sites that can be different or the same;
HCVENV is an HCV envelope protein or part thereof;
a, b, c, d, e and f are 0 or 1,
Here, the recombinant nucleic acid according to claim 1, wherein A1 and / or A2 may be a part of PS1 and / or A3 and / or A4 may be a part of PS2.   The recombinant nucleic acid of claim 1 or 2, further comprising a regulatory element that allows expression of the protein in a eukaryotic host cell.   The recombinant nucleic acid according to any one of claims 1 to 3, wherein the avian lysozyme leader peptide CL has an amino acid sequence defined by SEQ ID NO: 1.   A has an amino acid sequence selected from SEQ ID NOs: 63-65, 70-72 and 74-82, PS has an amino acid sequence selected from SEQ ID NOs: 66-68 and 83-84, or PS is Lys- A dibasic site such as Lys, Arg-Arg, Lys-Arg and Arg-Lys or a monobasic site such as Lys, wherein HCVENV is selected from SEQ ID NOs: 85-98 and fragments thereof. The recombinant nucleic acid according to 2 or 3.   A vector comprising the recombinant nucleic acid according to any one of claims 1 to 5.   The vector according to claim 6, which is an expression vector.   The vector according to claim 6 or 7, which is an autonomously replicating vector or an integrating vector.   The vector according to any one of claims 6 to 8, which is selected from SEQ ID NOs: 20, 21, 32, 35, 36, 39, 40, 49 and 50.   A host cell comprising the recombinant nucleic acid according to any one of claims 1 to 5 or the vector according to any one of claims 6 to 9.   11. The host cell of claim 10, which has the ability to express a protein comprising an avian lysozyme leader peptide or a functional equivalent thereof bound to an HCV envelope protein or a portion thereof. CL-[(A1) _a- (PS1) _b- (A2) _c ] -HCVENV-[(A3) _d- (PS2) _e- (A4) _f ]
Characterized by the structure;
In the formula;
CL is an avian lysozyme leader peptide or a functional equivalent thereof;
A1, A2, A3 and A4 are adapter peptides that can be different or the same;
PS1 and PS2 are processing sites that can be different or the same;
HCVENV is an HCV envelope protein or part thereof;
a, b, c, d, e and f are 0 or 1,
Wherein A1 and / or A2 may be part of PS1 and / or A3 and / or A4 is capable of expressing a protein that may be part of PS2. Host cell. When the CL peptide is removed, the protein CL-[(A1) _a- (PS1) _b- (A2) _c ] -HCVENV-[(A3) _d- (PS2) _e- (A4) _f ] The host cell according to any one of claims 10 to 12, wherein the protein and the CL peptide have the ability to translocate
CL-[(A1) _a- (PS1) _b- (A2) _c ] -HCVENV-[(A3) _d- (PS2) _e- (A4) _f ]
It is characterized by the structure
In the formula;
CL is an avian lysozyme leader peptide or a functional equivalent thereof;
A1, A2, A3 and A4 are adapter peptides that can be different or the same;
PS1 and PS2 are processing sites that can be different or the same;
HCVENV is an HCV envelope protein or part thereof;
a, b, c, d, e and f are 0 or 1,
Wherein A1 and / or A2 may be part of PS1 and / or A3 and / or A4 is derived from a protein that may be part of PS2.   14. A host cell according to any one of claims 10 to 13, having the ability to process the processing sites PS1 and / or PS2 in the protein translocated to the endoplasmic reticulum.   14. A host cell according to any one of claims 10 to 13 having the ability to N-glycosylate the protein translocated to the endoplasmic reticulum.   15. A host cell according to claim 14, having the ability to N-glycosylate the protein translocated to the endoplasmic reticulum and processed at the sites PS1 and / or PS2.   The host cell according to any one of claims 10 to 16, which is a eukaryotic cell.   The host cell according to any one of claims 10 to 16, which is a fungal cell.   18. A host cell according to claim 17, which is a yeast cell.   Saccharomyces Serevisiae cells, Saccaromyces kluyveri cell, or Saccharomyces uvarum cells such Saccharomyces cells, Schizosaccharomyces pombe cells such Schizosaccharomyces cell, Kluyveromyces cells such as Kluyveromyces lactis cell, Yarrowia lipolytica cells such Yarrowia cells, Hansenula cells such as Hansenula polymorpha cells, Pichia Neuro such as Pichia cells, Aspergillus cells, Neurospora crassa cells such as pastoris cells pora cells, mutants cells derived from any such Schwanniomyces cells or these Schwanniomyces occidentalis cells, the host cell of claim 19.   A method for producing an HCV envelope protein or a part thereof in a host cell, wherein the recombinant nucleic acid according to any one of claims 1 to 5 or any one of claims 6 to 9. A method comprising the step of transforming the host cell with the vector of claim 1, wherein the host cell comprises an avian lysozyme leader peptide or a functional equivalent thereof bound to an HCV envelope protein or a portion thereof. A method that has the ability to express. A method for producing an HCV envelope protein or a part thereof in a host cell, wherein the recombinant nucleic acid according to any one of claims 1 to 5 or any one of claims 6 to 9. Transforming the host cell with a vector comprising the host cell comprising:
CL-[(A1) _a- (PS1) _b- (A2) _c ] -HCVENV-[(A3) _d- (PS2) _e- (A4) _f ]
It is characterized by the structure
In the formula;
CL is an avian lysozyme leader peptide or a functional equivalent thereof;
A1, A2, A3 and A4 are adapter peptides that can be different or the same;
PS1 and PS2 are processing sites that can be different or the same;
HCVENV is an HCV envelope protein or part thereof;
a, b, c, d, e and f are 0 or 1,
Wherein A1 and / or A2 may be part of PS1 and / or A3 and / or A4 is capable of expressing a protein that may be part of PS2. When the CL peptide is removed from the host cell, the protein CL-[(A1) _a- (PS1) _b- (A2) _c ] -HCVENV-[(A3) _d- (PS2) _e- (A4) _f ] Having the ability to translocate to the endoplasmic reticulum, the protein and the CL peptide
CL-[(A1) _a- (PS1) _b- (A2) _c ] -HCVENV-[(A3) _d- (PS2) _e- (A4) _f ]
It is characterized by the structure
In the formula;
CL is an avian lysozyme leader peptide or a functional equivalent thereof;
A1, A2, A3 and A4 are adapter peptides that can be different or the same;
PS1 and PS2 are processing sites that can be different or the same;
HCVENV is an HCV envelope protein or part thereof;
a, b, c, d, e and f are 0 or 1,
23. The method according to claim 21 or 22, wherein A1 and / or A2 is derived from a protein that may be part of PS1 and / or A3 and / or A4 may be part of PS2. The method described.   24. The method of any one of claims 21 to 23, wherein the host cell has the ability to process processing sites PS1 and / or PS2 within the protein translocated to the endoplasmic reticulum.   24. The method according to any one of claims 21 to 23, further comprising in vitro processing of the processing sites PS1 and / or PS2.   24. The method of any one of claims 21 to 23, wherein the host cell has the ability to N-glycosylate the protein translocated to the endoplasmic reticulum.   25. The method of claim 24, wherein the host cell is capable of N-glycosylation of the protein translocated to the endoplasmic reticulum and processed at the sites PS1 and / or PS2.   28. A method according to any one of claims 21 to 27, wherein the host cell is a eukaryotic cell.   28. A method according to any one of claims 21 to 27, wherein the host cell is a fungal cell.   28. A method according to any one of claims 21 to 27, wherein the host cell is a yeast cell.   Said host cell, Saccharomyces Serevisiae cells, Saccaromyces kluyveri cell, or Saccharomyces uvarum cells such Saccharomyces cells, Schizosaccharomyces pombe cells such Schizosaccharomyces cell, Kluyveromyces cells such as Kluyveromyces lactis cell, Yarrowia lipolytica cells such Yarrowia cells of Hansenula polymorpha cells Hansenula cells, Pichia cells such as Pichia pastoris cells, Aspergillus cells, Neurospora crassa cells Neurospora cell is a mutant cells derived from any such Schwanniomyces cells or these Schwanniomyces occidentalis cells, The method according to any one of claims 21 to 27 when.   28. A method according to any one of claims 21 to 27, further comprising culturing the host cell in a suitable medium to obtain expression of the protein.   35. The method of claim 32, further comprising isolating the expressed protein from or from the host cell culture.   34. The method of claim 33, wherein the isolation step comprises lysing the host cell in the presence of a chaotropic agent.   35. The method of claim 33 or 34, wherein the thiol group of cysteine in the isolated protein is chemically modified and the chemical modification is reversible or irreversible.   36. A method according to any one of claims 32-35, comprising heparin affinity chromatography.

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