KR101618767B1 - Effective leader sequences needed for high expression of heterologous proteins - Google Patents

Abstract

The present invention relates to a leader sequence for efficiently producing an exogenous protein and a method for efficiently producing an exogenous protein using the same. More specifically, the present invention relates to 75 leader sequences for expressing foreign proteins with high efficiency and expression vectors , A cell line transfected with the expression vector, a method for producing a recombinant foreign protein using the cell line, and a screening method capable of selecting an optimal leader sequence for high expression of an exogenous protein. The leader sequence, vector and cell line of the present invention can be useful for the production of various biopharmaceuticals.

 Leader sequence, foreign protein, soluble receptor, cytokine, human monoclonal antibody, humanized monoclonal antibody

Description

[0002] Effective leader sequences for producing high-efficiency foreign proteins are disclosed in < RTI ID = 0.0 >

The present invention relates to a leader sequence for producing an exogenous protein with high efficiency and a method for efficiently producing an exogenous protein using the leader sequence.

Production of a useful protein using a recombinant gene is essentially required such as a host cell, a target gene, a vector, a selection marker, a promoter, and a leader sequence. Among them, the leader sequence secretes the protein produced in the cell out of the cell. In general, various growth factors and cytokines have a secretory signal peptide (leader sequence) at the amino terminus of the protein to promote the extracellular secretion of the mature protein, and the immature protein polypeptide synthesized in the cytoplasm is postranslationally modified. After the process, it is transferred to the cell membrane by the leader sequence and secreted out of the cell in the form of mature protein. In many cases, the leader peptide and the mature protein are cleaved by the action of a specific signal peptidase, and the mature protein is released to the outside of the cell. Regarding the cellular extracellular secretion of this general protein, the type and function of the leader sequence are described in McGeoch et al. al ., Virus Research 3, 271, 1985) and Heijnet et al . ( Nucleic Acid Research , 14, 4683, 1986).

Prior art related to the leader sequence has been studied in a large number of leader sequences in E. coli and yeast. U.S. Patent No. 5,919,654 discloses a leader sequence gene isolated from yeast. Korean Patent Registration No. 283677 discloses a newly synthesized leader sequence based on the common features of leader sequences secreted from E. coli. Korean Patent Publication No. 2003-62854 discloses a secretory-type vector that secretes granulocyte-stimulating factors with high efficiency, including a complex leader sequence composed of a leader sequence derived from two yeasts, and US Patent No. 5,010,003 And US Pat. No. 5,013,652 disclose an invertase secretion signal and an acid phosphatase leader sequence as leader sequences for yeast, respectively.

As a prior art relating to the leader sequence in animal cells, Korean Patent Laid-Open Publication No. 2001-0105066 (Nov. 28, 2001) discloses a cell line expressing human erythropoietin with high efficiency using an AMV (alfalfa mosaic virus) RNA4 leader sequence. It has also been reported that secretion is promoted by substituting the leader sequence of other proteins (Blam et al ., Oncogene 3 (2): 129-136, 1988).

In the production of recombinant proteins, secretion of the synthesized useful protein into a culture fluid has several advantages. The over expressed protein can be made into a water-soluble active protein by secreting the cytoplasm before it becomes an insoluble aggregate in the cytoplasm, and it is easier to separate and purify the protein secreted out of the cytoplasm compared with the protein present in the cytoplasm .

 Accordingly, in order to stably produce a cell line highly expressing an exogenous protein, a vector into which a gene coding for 75 leader sequences is inserted is prepared, and various cell lines and various soluble receptors are expressed. As a result, And thus the present invention has been completed.

It is an object of the present invention to provide a leader sequence and a recombinant expression vector containing the leader sequence, which allow expression of a recombinant foreign protein at higher efficiency than existing expression systems.

The present invention provides a leader sequence peptide characterized by high-efficiency expression of a recombinant foreign protein.

The present invention also provides a polynucleotide encoding the leader sequence peptide.

The present invention also provides a recombinant expression vector comprising a polynucleotide encoding said leader sequence peptide.

The present invention also provides a method for producing the recombinant expression vector.

In addition, the present invention provides a recombinant cell line transformed with the recombinant expression vector.

The leader sequence of the present invention and the recombinant expression vector containing the same can mass-produce recombinant foreign proteins including cell surface soluble receptors, cytokines and humanized monoclonal antibodies at high frequencies in animal cells, And can be usefully used in the production of medicines.

Definition of Terms:

A leader sequence or leader peptide sequence refers to a sequence existing at the N-terminus of a protein that induces secretion of a protein into a cell, and is also referred to as a signal sequence.

Recombinant refers to the act of producing DNA sequences that do not exist naturally through artificial manipulation.

A heterologous protein refers to a protein of exogenous origin that is introduced into a host by recombinant technology, rather than a protein produced by the host itself.

Polynucleotide means an organic polymer composed of nucleotide monomers linked by a covalent bond, including RNA and DNA molecules.

Fc refers to a site that binds to a receptor as a constant region of an antibody.

The present invention provides a leader sequence peptide selected from the group consisting of SEQ ID NOS: 1 to 75, characterized in that the recombinant foreign protein is expressed with high efficiency.

The present invention also provides a polynucleotide encoding the leader sequence peptide.

It is preferable that the polynucleotide has a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 76 to 150.

The present invention also provides a recombinant expression vector comprising a polynucleotide.

The skeletal vector used in the production of the expression vector may be any of those which can be used as a mammalian expression vector, and may be any of the following: pcDNA (Invitrogen, USA), pCMV (BD Bioscience, USA), pEF (Invitrogen, USA) (Promega, USA), pCI-neo (Promega, USA), pTK-neo (Novagen, USA), pFLAG (Sigma, USA) and p3xFLAG (Sigma, USA) vectors. 1 vector is inserted into an origin of oriP replication originated from Epstein-Barr virus, and a human antibody Fc region is cloned into a multiple cloning site for convenience of purification and increase of half-life in the body, More preferably a vector capable of being produced in the form of a fusion protein, and most preferably, the pYW600 vector used in the present invention is not limited thereto.

The present invention also provides a method for producing the recombinant expression vector.

The manufacturing method includes the following steps

1) preparing a polynucleotide encoding a leader sequence peptide selected from the group consisting of SEQ ID NOS: 1 to 75; And 2) operably inserting the polynucleotide of step 1) produced after the promoter sequence in the framework vector.

The skeletal vector of step 2) may be any of those available as a mammalian expression vector, and may be any one of pcDNA (Invitrogen, USA), pCMV (BD Bioscience, USA), pEF (Invitrogen, USA) (Sigma, USA) and p3XFLAG (Sigma, USA) vectors, and any one of the above vectors may be used as a vector. It is more preferable that the vector is inserted with the origin of replication of oriP derived from Epstein-Barr virus, and further, the human antibody Fc region is further cloned into the multiple cloning site, so that the recombinant protein is in the form of a fusion protein with the Fc More preferably, the vector is a pYW600 vector used in the present invention, but the present invention is not limited thereto.

The recombinant expression vector may additionally comprise a polynucleotide encoding an exogenous protein operably linked to a polynucleotide encoding the leader sequence peptide. The foreign protein may be a growth factor, a soluble receptor, a cytokine, or a monoclonal antibody, but the present invention is not limited thereto. The growth factor may be a transforming growth factor (TGF-beta), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), nerve growth factor (NGF), neurotrophin, platelet- , Erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF-8), growth differentiation factor-9 (GDF9), acidic fibroblast growth factor (aFGF or FGF- (BFGF or FGF-2), epithelial growth factor (EGF), hepatocyte growth factor (HGF), bone morphogenic factor (BMP) or vascular endothelial growth factor (VEGF), but not limited thereto, Lt; RTI ID = 0.0 > G-protein < / RTI > related receptor, receptor tyrosine kinase, But not limited to, an IL-2, IL-2, IF, interleukin-10, interleukin-1, 1 family, interleukin-12 family, or chemokine, but is not limited thereto. The monoclonal antibody is preferably a human antibody or a humanized antibody, but is not limited thereto.

In addition, the present invention provides a transformed recombinant cell line prepared by transforming a host cell with the recombinant expression vector.

Any eukaryotic cell can be used as the cell line, but it is preferably an animal cell line, and the animal cell line is more preferably a mammalian cell line. It is more preferable that the mammalian cell line is any one selected from the group consisting of HEK293E cells, HEK293T cells, CHO (Chinese hamster ovary) cells, BHK (baby hamster kidney) cells, NIH-3T3 cells and COS-7. In particular, when the expression vector used contains an oriP origin of replication, it is most preferably an HEK293E cell capable of expressing an exogenous protein by maintaining the expression vector in an episome state using the replication origin. However, But is not limited to.

Further,

I) operably inserting a polynucleotide sequentially comprising the leader sequence and the exogenous protein after the promoter sequence of the framework vector to produce a recombinant expression vector;

Ii) transfecting the host cell with the recombinant expression vector to prepare a transfected host cell;

Iii) culturing the transfected host cell; And

And iv) recovering the exogenous protein from the culture supernatant.

PCR (Promega), pCI (Promega, USA), pCMV (BD Bioscience, USA), pEF (Invitrogen, USA) USA), pCI-neo (Promega, USA), pTK-neo (Novagen, USA), pFLAG (Sigma, USA), p3xFLAG More preferably the human antibody Fc region is additionally cloned into the multiple cloning site so that the recombinant protein can be produced in the form of a fusion protein with the Fc More preferably a vector, and most preferably a pYW600 vector used in the present invention, but the present invention is not limited thereto.

In one embodiment of the present invention, the exogenous protein is preferably, but not limited to, a growth factor, a soluble receptor, a cytokine, or a monoclonal antibody. The growth factor may be selected from the group consisting of TGF-beta, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), nerve growth factor (NGF), neurotrophin neurotrophin, platelet derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF-8), growth differentiation factor-9 (GDF9), acid fibroblast growth factor (FGF-1), basic fibroblast growth factor (bFGF or FGF-2), epithelial growth factor (EGF), hepatocyte growth factor (HGF), bone morphogenic factor (BMP) or vascular endothelial growth factor But is not limited thereto. Preferably, the soluble receptor is a G-protein related receptor, a receptor tyrosine kinase or an ionotropic receptor, but is not limited thereto. The cytokine may be selected from the group consisting of interleukin-2 (IL-2), interferon, interleukin-10, interleukin-1 family, interleukin- But is not limited thereto. On the other hand, the monoclonal antibody is preferably a humanized antibody, but is not limited thereto.

In addition,

(I) A recombinant framework vector is prepared by inserting a polynucleotide encoding a human antibody Fc into a skeletal vector in a form capable of being expressed by a promoter of oriP originating from Epstein-Barr virus and a promoter of said skeletal vector step;

Ii) operatively linking a polynucleotide encoding an exogenous protein linked to the candidate leader sequence peptide to a polynucleotide encoding a human antibody Fc of said recombinant framework vector to produce a recombinant expression vector;

Iii) a control expression vector containing a polynucleotide encoding a fusion protein in which an indicator leader sequence is linked to the recombinant expression vector and the foreign protein in place of the candidate leader sequence peptide, respectively, is transfected into a host cell to prepare an experimental group and a control host cell ;

Iv) culturing the test group and the control host cells to recover respective culture supernatant;

V) quantifying the expression amount of an exogenous protein using an antibody specific for Fc; And

Vi) screening a leader sequence for high-throughput production of an exogenous protein, comprising the step of selecting a candidate leader sequence peptide that increases the production amount of the foreign protein in the test group as compared with the control group. In one embodiment of the invention, the exogenous protein is preferably a soluble receptor, cytokine or monoclonal antibody, more preferably a soluble receptor, most preferably soluble CD1D. In addition, the indicator leader sequence is preferably the reader sequence of the foreign protein itself, the alfalfa mosaic virus (AMV) RNA4 leader sequence, the yeast invertase secretion signal, and the yeast acid phosphatase leader sequence. However, Any leader sequence that has been used in protein production can be used. In addition, the recombinant expression vector preferably has a restriction enzyme cleavage site in front of the polynucleotide encoding the leader sequence for easy replacement of the leader sequence. The restriction enzyme is not particularly limited, It is preferably a lower restriction enzyme, more preferably BstEII. In the expression vector of the present invention, a restriction enzyme recognition site exists before and after a polynucleotide encoding an exogenous protein, and it is very easy to replace the foreign protein. The restriction enzyme recognition site is preferably not present in the original framework vector, but is not particularly limited thereto, and it is more preferable that it is SfiI.

The present inventors used the pcDNA3.1 vector (Invitrogen, USA) as a skeletal vector, and inserted an oriP replication origin on the CMV promoter of the pcDNA3.1 vector in order to increase the replication efficiency. Then, the immunoglobulin Fc domain Was inserted to prepare pcDNA3.1-oriP-Fc. Then, a polynucleotide encoding a recombinant protein to which a leader sequence (SEQ ID NO: 1) and a soluble CD1D (SEQ ID NO: 155) of a human antibody heavy chain (IGHV) gene having GeneBank accession number AAA51633 are linked is constructed by using a cDNA library prepared from human dendritic cells , And inserted into the Fc of the above-mentioned pcDNA3.1-oriP-Fc operably so as to be cloned, thereby preparing pYW600-CD1D.

Then, the present inventors have found that, in order to screen for a leader sequence efficiently expressing an exogenous protein in a water-soluble form, the leader sequence of SEQ ID NO: 1 is substituted with one leader sequence selected from the group consisting of SEQ ID NOS: 2 to 75 Work was performed. At this time, the BstEII site introduced when cloning the leader sequence of SEQ ID NO: 1 and the BstEII site located on the N-terminal side of the soluble domain of CD1D were used for the substitution.

The recombinant proteins composed of soluble CD1D as a total of 75 leader sequences and foreign proteins prepared as described above were introduced into the host cells and the production efficiency was increased (Table 3). In particular, SEQ ID NOS: 3, 6, 8, 19, 21, 44 and 45 were excellent (FIG. 2).

< Example  1> Preparation of recombinant expression vector with 75 leader sequences inserted

<1-1> Construction of skeletal vector

<1-1-1> Insertion of oriP

In order to clone oriP, a forward primer (5'-gt agatct gcaggaaaaggacaagc-3 ') shown in SEQ ID NO: 151 and a reverse primer (5'-cg agatct ggttgacttccctaatgt) described in SEQ ID NO: 152 were used as a template with pMEP4 (Invitrogen, USA) 3 ') was used for PCR reaction at 95 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 1 minute, and 30 cycles. PCR products of 2187 bp size were amplified by PCR purification kit (Promega, USA) . The PCR product was then digested with Bgl II enzyme (Invitrogen, USA) and the cleavage product was purified again. The purified cleaved fragment was digested with Bgl II enzyme (Invitrogen, USA) at the Bgl II recognition site present on the CMV promoter of pcDNA3.1 (Invitrogen, USA), inserted into a linearized vector, transformed into E. coli , And a transformant was obtained (pcDNA3.1-oriP). After the plasmid was extracted from the transformant by a conventional method, it was confirmed by sequencing that oriP was properly inserted.

&Lt; 1-1-2 > Insertion of polynucleotide encoding human antibody Fc

In order to insert the polynucleotide encoding the human antibody Fc into the pcDNA3.1 vector into which the oriP was inserted prepared in Example 1-1-1, a pLCN-MoH vector (Korean Patent No. 450266) was used as a template, (5'-cg gat cc g gcc gtg ggg gcc gac aaa a ct cacacatc c-3 ': SEQ ID NO: 153) consisting of the Bam HI-SfiI-human immunoglobulin Fc N-terminal sequence (SEQ ID NO: 154) was used as a primer (5'-c gagtc t ca ttt acc cgg aga cag gga-3 'composed of stop codon-human immunoglobulin Fc C-terminal sequence After the treatment, the treatment was repeated 30 times at 95 ° C for 30 seconds, at 60 ° C for 30 seconds, at 72 ° C for 1 minute, and then at 72 ° C for 10 minutes.

 The PCR products were then purified using PCR purification kit (Promega, USA). The purified PCR product was digested with restriction enzymes Bam HI and Xba I, and then ligated into pcDNA3.1 vector (pcDNA3.1 (SEQ ID NO: 2)) inserted with oriP prepared in Example 1-1-1, which was also digested with Bam HI and Xba I -oriP) to prepare pcDNA3.1-oriP-Fc. Thereafter, Escherichia coli (DH5?) Was transformed with the recombinant plasmid thus prepared, the transformant was isolated, and a plasmid was obtained to confirm that the polynucleotide encoding Fc was properly inserted.

<1-2> Insertion of a polynucleotide encoding an exogenous protein linked to a leader sequence

<1-2-1> Selection of candidate leader sequence

As candidate leader sequences, those listed in Table 1 below were selected.

Reader sequence information series
number SEQ ID NO: Gene symbol Protein reference number Leader amino acid sequence S00 One IGHV AAA51633 MGWSYIILFLVATATDVHS S01 2 INS P01308 MRLLPLLALLALWGPDPAAA SO2 3 PRSS1 P07477 MNPLLILTFVAAALA S03 4 ELA3B P08861 MMLRLLSSLLLVAVA S04 5 C1S P09871 MWCIVLFSLLAWVYA S05 6 ELA2B P08218 MIRTLLLSTLVAGALS S06 7 P4HB P07237 MLRRALLCLAVAALVRA S07 8 KLK3 P07288 MWVPVVFLTLSVTWIGA S08 9 ITIH3 NP_002208 MAFAWWPCLILALLSSLAAS S09 10 CLPS P04118 MEKILILLLVALSVAYA S10 11 PNLIPRP1 P54315 MLIFWTITLFLLGAAKG S11 12 AZGP1 P25311 MVPVLLSLLLLLGPAVP S12 13 PNLIPRP2 P54317 MLPPWTLGLLLLATVRG S13 14 C1R P00736 MWLLYLLVPALFCRAGG S14 15 SERPINF1 P36955 MQALVLLLCIGALLGHSSC S15 16 APOE P02649 MKVLWAALLVTFLAGCQA S16 17 APOA1 P02647 MKAAVLTLAVLFLTGSQA S17 18 ITIH2 P19823 MKRLTCFFICFFLSEVSG S18 19 CTRB1 P17538 MAFLWLLSCWALLGTTFG S19 20 KNG1 P01042 MKLITILFLCSRLLLSLT S20 21 HP P00738 MSALGAVIALLLWGQLFA S21 22 SAA4 P35542 MRLFTGIVFCSLVMGVTS S22 23 SAA1 P02735 MKLLTGLVFCSLVLGVSS S23 24 APOA2 P02652 MKLLAATVLLLTICSLEG S24 25 SEPP1 P49908 MWRSLGLALALCLLPSGGT S25 26 VH1-02 Antibody Heavy chain Gene MDWTWRILFLVAAATGAHS S26 27 VH1-46 Antibody Heavy chain Gene MDWTWRVFCLLAVAPGAHS S27 28 VH2-05 Antibody Heavy chain Gene MDTLCSTLLLLTIPSWVLS S28 29 VH3-11 Antibody Heavy chain Gene MEFGLSWVFLVAIIKGVQC S29 30 VH4-04 Antibody Heavy chain Gene MKHLWFFLLLVAAPRWVLS S30 31 VH5-51 Antibody Heavy chain Gene MGSTAILALLLAVLQGVCS S31 32 VH7-04 Antibody Heavy chain Gene MDWTWRILFLVAAATGAHS S32 33 APOH P02749 MISPVLILFSSFLCHVAIA S33 34 AMBP P02760 MRSLGALLLLLSACLAVSA S34 35 SERPIND1 P05546 MKHSLNALLIFLIITSAWG S35 36 FGA P02671 MFSMRIVCLVLSVVGTAWT S36 37 F12 P00748 MRALLLLGFLLVSLESTLS S37 38 DEFA6 Q01524 MRTLTILTAVLLVALQAKA S38 39 LPA /// PLG P08519 MEHKEVVLLLLLFLKSAAP S39 40 TF P02787 MRLAVGALLVCAVLGLCLA S40 41 VH6-01 Antibody Light chain Gene MSVSFLIFLPVLGLPWGVLS S41 42 VK-O11 Antibody Light chain Gene MRLPAQLLGLLMLWVPGSSE S42 43 VK-L2 Antibody Light chain Gene MEAPAQLLFLLLLLWLPDTTG S43 44 VK-B3 Antibody Light chain Gene MVLQTQVFISLLLWISGAYG S44 45 VK-B2 Antibody Light chain Gene MGSQVHLLSFLLLWISDTRA S45 46 GPX3 P22352 MARLLQASCLLSLLLAGFVS S46 47 APOC3 P02656 MQPRVLLVVALLALLASARA S47 48 APOA4 P06727 MFLKAVVLTLALVAVAGARA S48 49 TTR P02766 MASHRLLLLCLAGLVFVSEA S49 50 IGFBP5 P24593 MVLLTAVLLLAYAYAGPAQS S50 51 B2M P61769 MSRSVALAVLALLSLSGLEA S51 52 IGFBP4 P22692 MLPLCLVAALLLAAGPGPSLG S52 53 TFF3 Q07654 MAARALCMLGLVLALLSSSSA S53 54 GUCA2A Q02747 MNAFLLFALCLLGAWAALAGG S54 55 VK-A30 Antibody Light chain Gene MDMRVPAQLLGLLLLWFPGARC S55 56 APOC2 P02655 MGTRLLPALFLVLLVLGFEVQG S56 57 CLU P10909 MMKTLLLFVGLLLTWESGQVLG S57 58 PTGDS P41222 MATHHTLWMGLALLGVLGDLQA S58 59 C3 P01024 MGPTSGPSLLLLLLTHLPLALG S59 60 F9 P00740 MIMAESPGLITICLLGYLLSAEC S60 61 A2M P01023 MGKNKLLHPSLVLLLLVLLPTDA S61 62 SERPINA3 P01011 MERMLPLLALGLLAAGFCPAVLCLC S62 63 SERPINA1 P01009 MPSSVSWGILLLAGLCCLVPVSLA S63 64 AGT P01019 MAPAGVSLRATILCLLAWAGLAAG S64 65 BF P00751 MGSNLSPQLCLMPFILGLLSGGVTT S65 66 IGFBP7 Q16270 MERPSLRALLLGAAGLLLLLLPLSSS S66 67 APOC1 P02654 MRLFLSLPVLVVVLSIVLEGPAPAQG S67 68 GSN P06396 MAPHRPAPALLCALSLALCALSLPVRA S68 69 APOB P04114 MDPPRPALLALLALPALLLLLLAGARA S69 70 IGFBP3 P17936 MQRARPTLWAAALTLLVLLRGPPVARA S70 71 ITIH1 P19827 MDGAMGPRGLLLCMYLVSLLILQAMPA S71 72 FGB P02675 MVSWSFHKLKTMKHLLLLLLCVFLVKS S72 73 ITIH4 Q14624 MKPPRPVRTCSKVLVLLSLLAIHQTTTA S73 74 RNASE1 P07998 MALEKSLVRLLLLVLILLVLGWVQPSLG S74 75 FN1 P02751 MLRGPGPGLLLLAVQCLGTAVPSTGASKSKR

The sequence information of the polynucleotide encoding the leader sequence is shown in Table 2 below.

A nucleic acid sequence encoding a leader sequence series
number SEQ ID NO: Gene symbol GeneBank
Registration Number Nucleic acid sequence S00 76 IGHV K02149 atgggatggagctatatcatcctctttttggtagcaacagctacagatgtccactcc S01 77 INS NM_000207 atgcgcctcctgcccctgctggcgctgctggccctctggggacctgacccagccgcagcc SO2 78 PRSS1 NM_002770 atgaatctacttctgatccttacctttgttgcagctgctgttgct S03 79 ELA3B NM_007352 atgatgctccggctgctcagttccctcctccttgtggccgttgcc S04 80 C1S NM_001734 atgtggtgcattgtcctgttttcacttttggcatgggtttatgct S05 81 ELA2B NM_015849 atgattaggaccctgctgctgtccactttggtggctggagccctcagt S06 82 P4HB NM_000918 atgctgcgccgcgctctgctgtgcctggccgtggccgccctggtgcgcgcc S07 83 KLK3 NM_001648 atgtgggtcccggttgtcttcctcaccctgtccgtgacgtggattggtgct S08 84 ITIH3 NM_002217 atggcatttgcatggtggccctgtctcatcttggctctgctctccagcttggcagcctct S09 85 CLPS NM_001832 atggagaagatcctgatcctcctgcttgtcgccctctctgtggcctatgca S10 86 PNLIPRP1 NM_006229 atgctgatcttctggacaatcacacttttcctgctgggagcagccaaagga S11 87 AZGP1 NM_001185 atggtgcctgtcctgctgtctctgctgctgcttctgggtcctgctgtcccc S12 88 PNLIPRP2 NM_005396 atgctgcccccttggaccctcggccttctcctgctggccacagtcagagga S13 89 C1R NM_001733 atgtggctcttgtacctcctggtgccggccctgttctgcagggcaggaggc S14 90 SERPINF1 NM_002615 atgcaggccctggtgctactcctctgcattggagccctcctcgggcacagcagc S15 91 APOE NM_000041 atgaaggttctgtgggctgcgttgctggtcacattcctggcaggatgccaggcc S16 92 APOA1 NM_000039 atgaaagctgcggtgctgaccttggccgtgctcttcctgacggggagccaggct S17 93 ITIH2 NM_002216 atgaaaagactcacgtgctttttcatctgcttctttctttctgaagtatcaggc S18 94 CTRB1 NM_001025200 atggctttcctctggctcctctcctgctgggccctcctgggtaccaccttcggc S19 95 KNG1 NM_000893 atgaaactaattaccatccttttcctctgctccaggctgctactaagtttaacc S20 96 HP NM_005143 atgagtgccctgggagctgtcattgccctcctgctctggggacagctttttgca S21 97 SAA4 NM_006512 atgaggcttttcacaggcattgttttctgctccttggtcatgggagtcaccagt S22 98 SAA1 NM_000331 atgaagcttctcacgggcctggttttctgctccttggtcctgggtgtcagcagc S23 99 APOA2 NM_001643 atgaagctgctcgcagcaactgtgctactcctcaccatctgcagccttgaagga S24 100 SEPP1 NM_005410 atgtggagaagcctggggcttgccctggctctctgtctcctcccatcgggaggaaca S25 101 VH1-02 Antibody Heavy chain Gene atggactggacctggaggatcctcttcttggtggcagcagccacaggagcccactcc
S26 102 VH1-46 Antibody Heavy chain Gene atggactggacctggagggtcttctgcttgctggctgtagctccaggagctcactcc S27 103 VH2-05 Antibody Heavy chain Gene atggacacactttgctccacgctcctgctgctgaccatcccttcatgggtcttgtcc S28 104 VH3-11 Antibody Heavy chain Gene atggaatttgggctgagctgggttttccttgttgctattataaaaggtgtccagtgt S29 105 VH4-04 Antibody Heavy chain Gene atggaatttgggctgagctgggttttccttgttgctattataaaaggtgtccagtgt S30 106 VH5-51 Antibody Heavy chain Gene atggggtcaaccgccatcctcgccctcctcctggctgttctccaaggagtctgttcc S31 107 VH7-04 Antibody Heavy chain Gene atggactggacctggaggatcctcttcttggtggcagcagcaacaggtgcccactcc S32 108 APOH NM_000042 atgatttctccagtgctcatcttgttctcgagttttctctgccatgttgctattgca S33 109 AMBP NM_001633 atgaggagcctcggggccctgctcttgctgctgagcgcctgcctggcggtgagcgct S34 110 SERPIND1 NM_000185 atgaaacactcattaaacgcacttctcattttcctcatcataacatctgcgtggggt S35 111 FGA NM_000508 atgttttccatgaggatcgtctgcctggtcctaagtgtggtgggcacagcatggact S36 112 F12 NM_000505 atgagggctctgctgctcctggggttcctgctggtgagcttggagtcaacactttcg S37 113 DEFA6 NM_001926 atgagaaccctcaccatcctcactgctgttctcctcgtggccctccaggccaaggct S38 114 LPA /// PLG NM_005577 atggaacataaggaagtggttcttctacttcttttatttctgaaatcagcagcacct S39 115 TF NM_001063 atgaggctcgccgtgggagccctgctggtctgcgccgtcctggggctgtgtctggct S40 116 VH6-01 Antibody Light chain Gene atgtctgtctccttcctcatcttcctgcccgtgctgggcctcccatggggtgtcctgtca S41 117 VK-O11 Antibody Light chain Gene atgaggctccctgctcagctcctggggctgctaatgctctgggtccctggatccagtgag S42 118 VK-L2 Antibody Light chain Gene atggaagccccagcgcagcttctcttcctcctgctactctggctctcagataccaccgga S43 119 VK-B3 Antibody Light chain Gene atggtgttgcagacccaggtcttcatttctctgttgctctggatctctggtgcctacggg S44 120 VK-B2 Antibody Light chain Gene atggggtcccaggttcacctcctcagcttcctcctcctttggatctctgataccagggca S45 121 GPX3 NM_002084 atggcccggctgctgcaggcgtcctgcctgctttccctgctcctggccggcttcgtctcg S46 122 APOC3 NM_000040 atgcagccccgggtactccttgttgttgccctcctggcgctcctggcctctgcccgagct S47 123 APOA4 NM_000482 atgttcctgaaggccgtggtcctgaccctggccctggtggctgtcgccggagccagggct S48 124 TTR NM_000371 atggcttctcatcgtctgctcctcctctgccttgctggactggtatttgtgtctgaggct S49 125 IGFBP5 NM_000599 atggtgttgctcaccgcggtcctcctgctgctggccgcctatgcggggccggcccagagc S50 126 B2M NM_004048 atgtctcgctccgtggccttagctgtgctcgcgctactctctctctctggcctggaggct S51 127 IGFBP4 NM_001552 atgctgcccctctgcctcgtggccgccctgctgctggccgccgggcccgggccgagcctgggc S52 128 TFF3 L15203 atggctgccagagcgctctgcatgctggggctggtcctggccttgctgtcctcca gctctgct S53 129 GUCA2A M97496 atgaatgccttcctgctcttcgcactgtgcctccttggggcctgggccgccttggcaggaggg S54 130 VK-A30 Antibody Light chain Gene atggacatgagggtccccgctcagctcctggggctcctgctactctggttcccaggtgccagatgt S55 131 APOC2 NM_000483 atgggcacacgactcctcccagctctgtttcttgtcctcctggtattgggatttgaggtccagggg S56 132 CLU M64722 atgatgaagactctgctgctgtttgtggggctgctgctgacctgggagagtgggcaggtcctgggg S57 133 PTGDS NM_000954 atggctactcatcacacgctgtggatgggactggccctgctggggtgctgggcg acctgcaggca S58 134 C3 NM_000064 atgggacccacctcaggtcccagcctgctgctcctgctactaacccacctccccctggctctgggg S59 135 F9 NM_000133 atgatcatggcagaatcaccaggcctcatcaccatctgccttttaggatatctactcagtgctgaatgt S60 136 A2M M11313 atggggaagaacaaactccttcatccaagtctggttcttctcctcttggtcctcctgcccacagacgcc S61 137 SERPINA3 NM_001085 atggagagaatgttacctctcctggctctggggctcttggcggctgggttctgccctgctgtcctctgc S62 138 SERPINA1 BX161449 atgccgtcttctgtctcgtggggcatcctcctgctggcaggcctgtgctgcctggtccctgtctccctg S63 139 AGT NM_000029 atggctcctgccggtgtgagcctgagggccaccatcctctgcctcctggcctggg ctggcctggctgcaggt S64 140 BF NM_001710 atggggagcaatctcagcccccaactctgcctgatgccctttatcttgggcctcttgtctggaggtgtgaccacc S65 141 IGFBP7 NM_001553 atggagcggccgtcgctgcgcgccctgctcctcggcgccgctgggctgctgctcctgctcctgcccctctcctcttcc S66 142 APOC1 NM_001645 atgaggctcttcctgtcgctcccggtcctggtggtggttctgtcgatcgtcttggaaggcccagccccagcccagggg S67 143 GSN NM_000177 atggctccgcaccgccccgcgcccgcgctgctttgcgcgctgtccctggcgctgtgcgcgctgtcgctgcccgtccgcgcg S68 144 APOB NM_000384 atggacccgccgaggcccgcgctgctggcgctgctggcgctgcctgcgctgctgctgctgctgctggcgggcgccagg S69 145 IGFBP3 NM_000598 atgcagcgggcgcgacccacgctctgggccgctgcgctgactctgctggtgctgctccgcgggccgccggtggcgcgggct S70 146 ITIH1 NM_002215 atggacggtgccatggggcctcgggggctgctgttgtgcatgtacctggtatctctcctcatcctgcaggccatgcctgcc S71 147 FGB NM_005141 atggtttcttggagcttccacaaacttaaaaccatgaaacatctattattgctactattgtgtgtttttctagttaagtcc S72 148 ITIH4 NM_002218 atgaagcccccaaggcctgtccgtacctgcagcaaagttctcgtcctgctttcactgctggccatccaccagactactactcc S73 149 RNASE1 NM_002933 atggctctggagaagtctcttgtccggctccttctgcttgtcctgatactgctggtgctgggctgggtccagccttccctgggc S74 150 FN1 NM_002026 atgcttaggggtccggggcccgggctgctgctgctggccgtccagtgcctggggacagcggtgccctccacgggagcctcgaagagcaagagg

&Lt; 1-2-2 > Cloning of polynucleotide encoding exogenous protein linked to leader sequence

In order to insert the leader sequence shown in Table 1 and the polynucleotide encoding the CD1D fusion protein linked thereto in frame, into the skeleton vector prepared in Example 1-1, The gene encoding the fusion protein of the soluble receptor (SEQ ID NO: 155) of the CD1D protein was first cloned into the framework vector. Specifically, a cDNA library prepared from human dendritic cells was used as a template, and a forward primer (5'-caggg ggcc gtg ggg gcc gtc ccg caa agg ctt ttc cc-3 composed of SfiI-CD1D N-terminal shown in SEQ ID NO: 156 (5'-ta gc g gcc ggg ggg gcc gga ggt gta gct ccc acc-3 ') consisting of the Sfi-CD1D C-terminal of SEQ ID NO: The PCR product was purified after the PCR reaction was performed under the conditions of 30 sec. For 72 sec. For 1 min. And 30 cycles. Then, the PCR product was digested with Sfi I, Vector to prepare pcDNA3.1-oriP-CD1D-Fc. Then, to clone the leader sequence of protein sequence number AAA51633, which is an immunoglobulin heavy chain, the forward primer of SEQ ID NO: 158 and the reverse primer of SEQ ID NO: 154 described in the following Table 2 were used to clone the pcDNA3.1- PCR was carried out using oriP-CD1D-Fc as a template. Subsequently, the PCR reaction product was digested with Nhe I and Xba I and inserted into pcDNA3.1-oriP-CD1D-Fc digested with Nhe I and Xba I to prepare pcDNA3.1-oriP-S00-CD1D-Fc And named pYW600-CD1D. Next, in order to replace S00 in S01 to S74 described in Table 2, the forward primer described in SEQ ID Nos. 158 to 256 described in the following Table 3 and the reverse primer (5'-ttggcgaattccttcacgtccctggtgaagctgcttcgggtatacccgaa-3 ') described in SEQ ID NO: 257 The PCR product was purified using the above pYW600-CD1D as a template at 95 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 1 minute, and 30 cycles. The PCR product was purified and the BstEII enzyme (NEB, USA) And then inserted into pYW600-CD1D, which was also digested with BstEII enzyme to prepare a recombinant vector. Then, the E. coli host cell DH5? (Invitrogen, USA) was transformed with the recombinant vector. In the case of SEQ ID NOS: 158 to 208 corresponding to the leader sequences S00 to S50, the 5'-terminal of the forward primer is a nucleic acid sequence coding for the leader sequence and the 3'- Is the sequence corresponding to the N-terminus of the water-soluble CD1D to clone a water-soluble CD1D protein that is properly ligated. On the other hand, in the case of the leader sequences S51 to S74, since the leader sequence itself is long, a single forward primer can not be used and the forward primer is substituted with A (SEQ ID NOS: 209 to 232) and B (SEQ ID NOS: 233 to 256) And the gene cassette was prepared through two successive PCR reactions. A plasmid was obtained from the isolated transformant, and a clone into which the gene encoding the fusion protein in the first direction was inserted was selected.

The primers used for the leader sequence cloning primer SEQ ID NO: order S00 158 gg gctagcggtcacc gccacc
atg gga tgg agc tat atc atc ctc ttt ttg gta gca aca gct aca gat gtc cac tcc cag gg g gcc gtg ggg S01 159 ctagc ggtcacc gccacc
atg cgc ctc ctc ctg ctg gcg ctg ctg gcc ctc tgg gga cct gac cca gcc gca gcc gg g gcc gtg ggg g SO2 160 ctagc ggtcacc gccacc
atg aat cta ctt ctg atc ctt acc ttt gtt gca gct gct gtt gct gct gg g gcc gtg ggg g S03 161 ctagc ggtcacc gccacc
atg atg ctc cgg ctg ctc agt tcc ctc ctc ctt gtg gcc gtt gcc tct gg g gcc gtg ggg g S04 162 ctagc ggtcacc gccacc
atg tgg tgc att gtc ctg ttt tca ctt ttg gca tgg gtt tat gct gaa gg g gcc gtg ggg g S05 163 ctagc ggtcacc gccacc
atg att agg acc ctg ctg ctg tcc act ttg gtg gct gga gcc ctc agt gaa gg g gcc gtg ggg g S06 164 ctagc ggtcacc gccacc
atg ctg cgc cgc gct ctg ctg tgc ctg gcc gtg gcc gcc ctg gtg cgc gcc gac gg g gcc gtg ggg g S07 165 ctagc ggtcacc gccacc
atg tgg gtc ccg gtt gtc ttc ctc acc ctg tcc gtg acg tgg att ggt gct gct gg g gcc gtg ggg g S08 166 ctagc ggtcacc gccacc
atg gca ttt gca tgg tgg ccc tgt ctc atc ttg gct ctg ctc tcc agc ttg gca gcc tct gg g gcc gtg ggg g S09 167 ctagc ggtcacc gccacc
atg gag aag atc ctg atc ctc ctg ctt gtc gcc ctc tct gtg gcc tat gca gca gg g gcc gtg ggg g S10 168 ctagc ggtcacc gccacc
atg ctg atc ttc tgg aca atc aca ctt ttc ctg ctg gga gca gcc aaa gga aaa gg g gcc gtg ggg g S11 169 ctagc ggtcacc gccacc atg gtg cct gtc ctg ctg tct ctg ctg ctg ctt ctg ggt cct gct gtc ccc cag gg g gcc gtg ggg g S12 170 ctagc ggtcacc gccacc
atg ctg ccc cct tgg acc ctc ggc ctt ctc ctg ctg gcc aca gtc aga gga aaa gg g gcc gtg ggg g S13 171 ctagc ggtcacc gccacc
atg tgg ctc ttg tac ctc ctg gtg ccg gcc ctg ttc tgc agg gca gga ggc tct gg g gcc gtg ggg g S14 172 ctagc ggtcacc gccacc
atg cag gcc ctg gtg ct ctc ctc tgc att gga gcc ctc ctc ggg cac agc agc cag gg g gcc gtg ggg g S15 173 ctagc ggtcacc gccacc
atg aag gtt ctg tgg gct gcg ttg ctg gtc aca ttc ctg gca gga tgc cag gcc aaa gg g gcc gtg ggg g S16 174 ctagc ggtcacc gccacc
atg aaa gct gcg gtg ctg acc ttg gcc gtg ctc ttc ctg acg ggg agc cag gct cga gg g gcc gtg ggg g S17 175 ctagc ggtcacc gccacc
atg aaa aga ctc acg tgc ttt ttc atc tgc ttc ttt ctt tct gaa gta tca ggc ttc gg g gcc gtg ggg g S18 176 ctagc ggtcacc gccacc
atg gct ttc ctc tgg ctc ctc tcc tgc tgg gcc ctc ctg ggt acc acc ttc ggc aaa gg g gcc gtg ggg g S19 177 ctagc ggtcacc gccacc
atg aaa cta att acc atc ctt ttc ctc tgc tcc agg ctg cta cta agt tta acc cag gg g gcc gtg ggg g S20 178 ctagc ggtcacc gccacc
atg agt gcc ctg gga gct gtc att gcc ctc ctg ctc tgg gga cag ctt ttt gca gtg gg g gcc gtg ggg g S21 179 ctagc ggtcacc gccacc
atg agg ctt ttc aca ggc att gtt ttc tgc tcc ttg gtc atg gga gtc acc agt gaa gg g gcc gtg ggg g S22 180 ctagc ggtcacc gccacc
atg aag ctt ctc acg ggc ctg gtt ttc tgc tcc ttg gtc ctg ggt gtc agc agc cga gg g gcc gtg ggg g S23 181 ctagc ggtcacc gccacc
atg aag ctg ctc gca gca act gtg cta ctc ctc acc atc tgc agc ctt gaa gga gca gg g gcc gtg ggg g S24 182 ctagc ggtcacc gccacc
atg tgg aga agc ctg ggg ctt gcc ctg gct ctc tgt ctc ctc cca tcg gga gga aca gaa gg g gcc gtg ggg g S25 183 ctagc ggtcacc gccacc
atg gac tgg acc tgg agg atc ctc ttc ttg gtg gca gca gcc aca gga gcc cac tcc cag gg g gcc gtg ggg g S26 184 ctagc ggtcacc gccacc
atg gac tgg acc tgg agg gtc ttc tgc ttg ctg gct gta gct cca gga gct cac tcc cag gg g gcc gtg ggg g S27 185 ctagc ggtcacc gccacc
atg gac aca ctt tgc tcc acg ctc ctg ctg ctg acc atc cct tca tgg gtc ttg tcc cag gg g gcc gtg ggg g S28 186 ctagc ggtcacc gccacc
ttt ggg agc ctg tgg gtt gaa atg ttc ctt gtt gct att ata aaa ggt gtc cag tgt cag gg g gcc gtg ggg g S29 187 ctagc ggtcacc gccacc
cac ctg tgg ctc gaa ttc ttc atg ctg ctg gtg gct gca ccc aga ctg tgg gtc tcc cag gg g gcc gtg ggg g S30 188 ctagc ggtcacc gccacc
ggg tca atc ctc gcc gcc acc atg ctc ctc ctg gct gtt ctc caa gga gtc tgt tcc gag gg g gcc gtg ggg g S31 189 ctagc ggtcacc gccacc
atg gac tgg acc tgg agg atc ctc ttc ttg gtg gca gca gca aca ggt gcc cac tcc cag gg g gcc gtg ggg g S32 190 ctagc ggtcacc gccacc
atg att tct cc gtg ctc atc ttg ttc tcg agt ttt ctc tgc cat gtt gct att gca gg g gcc gtg ggg g S33 191 ctagc ggtcacc gccacc
atg agg agc ctc ggg gcc ctg ctc ttg ctg ctg agc gcc tgc ctg gcg gtg agc gct gg g gcc gtg ggg g S34 192 ctagc ggtcacc gccacc
atg aaa cac tca tta aac gca ctt ctc att ttc ctc atc ata aca tct gcg tgg ggt gg g gcc gtg ggg g S35 193 ctagc ggtcacc gccacc
atg ttt tcc atg agg atc gtc tgc ctg gtc cta agt gtg gtg ggc aca gca tgg act gca gg g gcc gtg ggg g S36 194 ctagc ggtcacc gccacc
atg agg gct ctg ctg ctc ctg ggg ttc ctg ctg gtg agc ttg gag tca aca ctt tcg atc gg g gcc gtg ggg g S37 195 ctagc ggtcacc gccacc
atg aga acc ctc acc atc ctc act gct gtt ctc ctc gtg gcc ctc cag gcc aag gct gaa gg g gcc gtg ggg g S38 196 ctagc ggtcacc gccacc
atg gaa cat aag gaa gta gta ct cta ctt ctt tta ttt ctg aaa tca gca gca cct gaa gg g gcc gtg ggg g S39 197 ctagc ggtcacc gccacc
atg agg ctc gcc gtg gga gcc ctg ctg gtc tgc gcc gtc ctg ggg ctg tgt ctg gct gtg gg g gcc gtg ggg g S40 198 ctagc ggtcacc gccacc
atg tct gtc tcc ttc ctc atc ttc ctg ccc gtg ctg ggc ctc cca tgg ggt gtc ctg tca cag g gg gcc gtg ggg g S41 199 ctagc ggtcacc gccacc
atg agg ctc cct gct cag ctc ctg ggg ctg cta atg ctc tgg gtc cct gga tcc agt gag gat gg g gcc gtg ggg g S42 200 ctagc ggtcacc gccacc
atg gaa gcc cca gcg cag ctt ctc ttc ctc ctg cta ctc tgg ctc cca gat acc acc gga gaa gg g gcc gtg ggg g S43 201 ctagc ggtcacc gccacc
atg gtg ttg cag acc cag gtc ttc att tct ctg ttg ctc tgg atc tct ggt gcc tac ggg gac gg g gcc gtg ggg g S44 202 ctagc ggtcacc gccacc
atg ggg tcc cag gtt cac ctc ctc agc ttc ctc ctc ctt tgg atc tct gat acc agg gca gaa gg g gcc gtg ggg g S45 203 ctagc ggtcacc gccacc
atg gcc cgg ctg cgg gcg tcc tgc ctg ctt tcc ctg ctc ctg gcc ggc ttc gtc tcg cag ggg gcc gtg ggg g S46 204 ctagc ggtcacc gccacc
ccc cgg gta gtt ctc ctt cag atg gtt gcc ctc ctc ctg ctg gcg gcc gcc tct cga gct tct gg g gcc gtg ggg g S47 205 ctagc ggtcacc gccacc
atg ttc ctg aag gcc gtg gtc ctg acc ctg gcc ctg gtg gct gtc gcc gga gcc agg gct gaa ggg gcc gtg ggg g S48 206 ctagc ggtcacc gccacc
atg gct tct cat cgt ctg ctc ctc ctc tgc ctt gct gga ctg gta ttt gtg tct gag gct gg g gcc gtg ggg g S49 207 ctagc ggtcacc gccacc
atg gtg ttg ctc acc gcg gtc ctc ctg ctg ctg gcc gcc tat gcg ggg ccg gcc cag agc ctg ggg gcc gtg ggg g S50 208 ctagc ggtcacc gccacc
atg tct cgc tcc gtg gcc tta gct gtg ctc gcg cta ctc tct ctt tct ggc ctg gag gct atc gg g gcc gtg ggg g S51A 209 ctagc ggtcacc gccacc
atg gtg ccc ctc tgc ctc gtg gcc gcc ctg ctg ctg gcc gcc ggg cc S52A 210 ctagc ggtcacc gccacc
atg gct gcc aga gcg ctc tgc atg ctg ggg ctg gtc ctg gcc ttg ct S53A 211 ctagc ggtcacc gccacc
atg aat gcc ttc ctg ctc ttc gca ctg tgc ctc ctt ggg gcc tgg gc S54A 212 ctagc ggtcacc gccacc
atg gac atg agg gtc ccc gct cag ctc ctg ggg ctc ctg cta ctc tg S55A 213 ctagc ggtcacc gccacc
atg ggc aca cga ctc ctc cca gct ctg ttt ctt gtc ctc ctg gta tt S56A 214 ctagc ggtcacc gccacc
atg ag act ctg ctg ctg ttt ggg cgg ctg ctg tgg ga S57A 215 ctagc ggtcacc gccacc
atg gct act cat cac acg ctg tgg atg gga ctg gcc ctg ctg ggg gt S58A 216 ctagc ggtcacc gccacc
atg gga ccc acc tca ggt ccc agc ctg ctg ctc ctg cta cta acc ca S59A 217 ctagc ggtcacc gccacc
atg atc gc gaa tca cca ggc ctc atc acc atc tgc ctt tta gg S60A 218 ctagc ggtcacc gccacc
atg ggg aag aac aaa ctc ctt cat cca agt ctg gtt ctt ctc ctc tt S61A 219 ctagc ggtcacc gccacc
atg gag aga atg tta cct ctc ctg gct ctg ggg ctc ttg gcg gct gg S62A 220 ctagc ggtcacc gccacc
atg ccg tct tct gtc tcg tgg ggc atc ctc ctg ctg gca ggc ctg tg S63A 221 ctagc ggtcacc gccacc
atg gct cct gcc ggt gtg agc ctg agg gcc acc atc ctc tgc ctc ct S64A 222 ctagc ggtcacc gccacc
atg ggg agc aat ctc agc ccc caa ctc tgc ctg atg ccc ttt atc tt S65A 223 ctagc ggtcacc gccacc
atg gag cgc gcg tcg ctg cgc gcc ctg ctc ttc ggc ccc gct ggg ct S66A 224 ctagc ggtcacc gccacc
atg agg ctc ttc ctg tcg ctc ccg gtc ctg gtg gtg gtt ctg tcg at S67A 225 ctagc ggtcacc gccacc
atg gct ccg cac cgc ccc gcg ccc gcg ctg ctt tgc gcg ctg tcc ct S68A 226 ctagc ggtcacc gccacc
atg gac ccg ccg agg ccc gcg ctg ctg gcg ctg ctg gcg ctg cct gc S69A 227 ctagcggtcaccgccacc
atg cag cgg gcg cga ccc acg ctc tgg gcc gct gcg ctg act ctg ct S70A 228 ctagc ggtcacc gccacc
atg gac ggt gcc atg ggg cct cgg ggg ctg ctg ttg tgc atg tac ct S71A 229 ctagc ggtcacc gccacc
atg gtt tct tgg agc ttc cac aaa ctt aaa acc atg aaa cat cta tt S72A 230 ctagc ggtcacc gccacc
atg aag ccc cca agg cct gtc cgt acc tgc agc aaa gtt ctc gtc ct S73A 231 ctagc ggtcacc gccacc
atg gct ctg gag aag tct ctt gtc cgg ctc ctt ctg ctt gtc ctg at S74A 232 ctagc ggtcacc gccacc
atg ctt agg ggt cgg ggg ccc ggg ctg ctg ctg ctg gcc gtc cag tgc ctg ggg ac S51B 233 gctgctggccgccgggcccgggccgagcctgggcgatgg ggccgtggggg S52B 234 gctggtcctggccttgctgtcctccagctctgctgaagg ggccgtggggg S53B 235 cctccttggggcctgggccgccttggcaggaggggtggg ggccgtggggg S54B 236 ggggctcctgctactctggttcccaggtgccagatgtgacgg ggccgtggggg S55B 237 tcttgtcctcctggtattgggatttgaggtccaggggaccgg ggccgtggggg S56B 238 gctgctgctgacctgggagagtgggcaggtcctgggggatgg ggccgtggggg S57B 239 actggccctgctgggggtgctgggcgacctgcaggcagcagg ggccgtggggg S58B 240 gctcctgctactaacccacctccccctggctctggggtctgg ggccgtggggg S59B 241 caccatctgccttttaggatatctactcagtgctgaatgtactgg ggccgtggggg S60B 242 tctggttcttctcctcttggtcctcctgcccacagacgcctctgg ggccgtggggg S61B 243 ggggctcttggcggctgggttctgccctgctgtcctctgccacgg ggccgtggggg S62B 244 cctgctggcaggcctgtgctgcctggtccctgtctccctggctgaagg ggccgtggggg S63B 245 caccatcctctgcctcctggcctgggctggcctggctgcaggtgatgg ggccgtggggg S64B 246 cctgatgccctttatcttgggcctcttgtctggaggtgtgaccaccactgg ggccgtg gggg S65B 247 cttcggccccgctgggctgctgctcctgctcctgcccctctcctcttcctctgg ggcc gtggggg S66B 248 ggtggtggttctgtcgatcgtcttggaaggcccagccccagcccaggggactgg ggcc gtggggg S67B 249 gctttgcgcgctgtccctggcgctgtgcgcgctgtcgctgcccgtccgcgcggcagg g gccgtggggg S68B 250 gctgctggcgctgcctgcgctgctgctgctgctgctggcgggcgccagggccgaagg g gccgtggggg S69B 251 cgctgcgctgactctgctggtgctgctccgcgggccgccggtggcgcgggctgg ggcc gtggggg S70B 252 gctgttgtgcatgtacctggtatctctcctcatcctgcaggccatgcctgccctggg g gccgtggggg S71B 253 aaccatgaaacatctattattgctactattgtgtgtttttctagttaagtcccaggg g gccgtggggg S72B 254 cagcaaagttctcgtcctgctttcactgctggccatccaccagaccactactgccgaagg ggccgtggggg S73B 255 ccttctgcttgtcctgatactgctggtgctgggctgggtccagccttccctgggcaaagg ggccgtggggg S74B 256 cgtccagtgcctgggacagcggtgccctccacgggagcctcgaagagcaagaggcaggg ggccgtggggg

In the sequence shown in Table 3, the underlined portion indicates the restriction enzyme recognition site, the underline at the 5'-end indicates the BstEII recognition site, the underline at the 3'-end indicates the SfiI recognition site, .

Example 2 Expression of Recombinant Expression Vector into CD1D, a Soluble Receptor Connected to 75 Leader Sequences

&Lt; 2-1 > Expression in HEK293E cells

Each of the recombinant vectors containing 75 different leader sequences prepared in Example <1-2> was transfected into HEK293E cells using PEI (polyethyleneimmune, Polysciences, USA). HEK293E cells transfected with each expression vector were cultured in serum-free medium containing DMEM, 50 占 퐂 / ml penicillin, and 50 占 퐂 / ml streptomycin. The cell culture was incubated at 37 ° C, 5% CO ₂ for 72 hours, and the culture supernatant was collected.

<2-2> Measurement of expression level

Transient levels of CD1D expression in cells transfected with each of the 75 expression vectors were examined by enzyme immunoassay as described above. Specifically, 100 μl of anti-human antibody Fc region-specific Fab antibody (Sigma, USA) was coated at a concentration of 1 μg / ml at room temperature for 1 hour in each of the designated wells of a 96-well plate. After the reaction, the remaining reaction solution of each well was removed and washed with washing solution three times with 250 쨉 l of washing solution. The culture medium of each of the 75 species collected in Example 2-1 was diluted by 1/5 and reacted at room temperature for 1 hour. At the same time, Embrel was used as a standard solution for quantification and reacted at a dilution ratio equal to that of the culture supernatant. After washing three times, HRP-labeled anti-human antibody Fc region specific antibody (Sigma, USA) was diluted at a ratio of 1: 1000 and reacted at room temperature for 1 hour. After the reaction solution was completely removed, the reaction solution was washed three times with washing solution, and 100 μl of the substrate-coloring solution was added thereto, followed by reaction at room temperature for 2 minutes. To each well, 100 반응 of each reaction stop solution was added and absorbance was measured at 450 nm.

As described above, the respective expression levels were measured by an ELISA assay, and the expression levels were as shown in Table 3 below. 605, 607, 618, 620, 643, 644 and the like exhibited higher expression levels than pYW602 (vector having CD1D inserted with the S02 leader sequence) and the original leader sequence S00.

The expression level of recombinant soluble receptor CD1D clone Expression level  ( ng / ml ) One day 3 day 5 day 600 -6 352 971 601 47 861 1757 602 -One 443 1093 603 4 773 1933 605 -21 345 1155 607 -6 456 1379 609 -12 323 913 610 -12 236 752 613 -21 143 468 615 -16 242 808 616 -3 302 727 617 -11 531 1480 618 -14 473 1248 620 -13 434 1315 621 -21 203 820 622 -20 178 697 623 -25 121 588 624 -27 106 401 625 -18 169 583 626 -29 -27 -9 627 -16 244 791 643 -17 456 1277 644 -21 339 1190 672 -3 254 737 646 -31 153 515 647 -13 203 690 648 -18 264 866 649 -21 244 713 650 -45 -32 -5 652 -37 112 455 653 -40 72 429 654 -36 174 681 655 -32 190 741 656 -33 146 606 662 -33 113 492 673 -33 242 860 663 -49 77 -8 665 -42 92 389 666 -39 82 429 667 -47 -38 -18 670 -22 250 852

<2-3> Western Blot

The 75 cultures collected in Example <2-1> were analyzed by Western blotting. The Western blot method is as follows. The culture samples were mixed with SDS sample buffer and 50 μg lysate and heated on a 10% SDS-PAGE gel. The separated proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk. Then, HRP-labeled anti-human antibody Fc region specific antibody (Sigma, USA) was diluted at a ratio of 1: 1000 and reacted at room temperature for 1 hour. The ECL kit (ECL Plus, Amersham, USA) was then processed according to the manufacturer's instructions.

As a result, it was possible to confirm the band at the same position as the expected protein size, and confirm the difference in the expression amount as shown in Example 2-2 (FIG. 2).

Figure 1 is a cleavage map of the pYW600-CD1D vector.

FIG. 2 shows the result of Western blot analysis of the expression level of soluble receptor CD1D after transfection of a vector containing some of the 75 prepared leader sequences in HEK293E cells.

                         SEQUENCE LISTING <110> Korea Research Institute of Bioscience and Biotechnology   <120> Effective leader sequences needed for high expression of soluble        receptors, cytokines and monoclonal antibodies <130> 7p-10-04 <160> 257 <170> PatentIn version 3.5 <210> 1 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> IGHV leader sequence <400> 1 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Asp 1 5 10 15 Val His Ser              <210> 2 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> INS leader sequence <400> 2 Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala Leu Trp Gly Pro Asp 1 5 10 15 Pro Ala Ala Ala             20 <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PRSS1 leader sequence <400> 3 Met Asn Pro Leu Leu Ile Leu Thr Phe Val 1 5 10 15 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> ELA3B leader sequence <400> 4 Met Met Leu Arg Leu Leu Ser Ser Leu Leu Leu Val Ala Val Ala 1 5 10 15 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> C1S leader sequence <400> 5 Met Trp Cys Ile Val Leu Phe Ser Leu Leu Ala Trp Val Tyr Ala 1 5 10 15 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> ELA2B leader sequence <400> 6 Met Ile Arg Thr Leu Leu Leu Ser Thr Leu Val Ala Gly Ala Leu Ser 1 5 10 15 <210> 7 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> P4HB leader sequence <400> 7 Met Leu Arg Arg Ala Leu Leu Cys Leu Ala Val Ala Ala Leu Val Arg 1 5 10 15 Ala      <210> 8 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> KLK3 leader sequence <400> 8 Met Trp Val Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile Gly 1 5 10 15 Ala      <210> 9 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> ITIH3 leader sequence <400> 9 Met Ala Phe Ala Trp Trp Pro Cys Leu Ile Leu Ala Leu Leu Ser Ser 1 5 10 15 Leu Ala Ala Ser             20 <210> 10 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CLPS leader sequence <400> 10 Met Glu Lys Ile Leu Ile Leu Leu Leu Val Ala Leu Ser Val Ala Tyr 1 5 10 15 Ala      <210> 11 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> PNLIPRP1 leader sequence <400> 11 Met Leu Ile Phe Trp Thr Ile Thr Leu Phe Leu Leu Gly Ala Ala Lys 1 5 10 15 Gly      <210> 12 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> AZGP1 leader sequence <400> 12 Met Val Pro Val Leu Leu Ser Leu Leu Leu Leu Leu Gly Pro Ala Val 1 5 10 15 Pro      <210> 13 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> PNLIPRP2 leader sequence <400> 13 Met Leu Pro Pro Trp Thr Leu Gly Leu Leu Leu Leu Ala Thr Val Arg 1 5 10 15 Gly      <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> C1R leader sequence <400> 14 Met Trp Leu Leu Tyr Leu Leu Val Pro Ala Leu Phe Cys Arg Ala Gly 1 5 10 15 Gly      <210> 15 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> SERPINF1 leader sequence <400> 15 Met Gln Ala Leu Val Leu Leu Leu Cys Ile Gly Ala Leu Leu Gly His 1 5 10 15 Ser Ser Cys              <210> 16 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> APOE leader sequence <400> 16 Met Lys Val Leu Trp Ala Ala Leu Leu Val Thr Phe Leu Ala Gly Cys 1 5 10 15 Gln Ala          <210> 17 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> APOA1 leader sequence <400> 17 Met Lys Ala Ala Val Leu Thr Leu Ala Val Leu Phe Leu Thr Gly Ser 1 5 10 15 Gln Ala          <210> 18 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> ITIH2 leader sequence <400> 18 Met Lys Arg Leu Thr Cys Phe Phe Ile Cys Phe Phe Leu Ser Glu Val 1 5 10 15 Ser Gly          <210> 19 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> CTRB1 leader sequence <400> 19 Met Ala Phe Leu Trp Leu Leu Ser Cys Trp Ala Leu Leu Gly Thr Thr 1 5 10 15 Phe Gly          <210> 20 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> KNG1 leader sequence <400> 20 Met Lys Leu Ile Thr Ile Leu Phe Leu Cys Ser Arg Leu Leu Leu Ser 1 5 10 15 Leu Thr          <210> 21 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> HP leader sequence <400> 21 Met Ser Ala Leu Gly Ala Val Ile Ala Leu Leu Leu Trp Gly Gln Leu 1 5 10 15 Phe Ala          <210> 22 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> SAA4 leader sequence <400> 22 Met Arg Leu Phe Thr Gly Ile Val Phe Cys Ser Leu Val Met Gly Val 1 5 10 15 Thr Ser          <210> 23 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> SAA1 leader sequence <400> 23 Met Lys Leu Leu Thr Gly Leu Val Phe Cys Ser Leu Val Leu Gly Val 1 5 10 15 Ser Ser          <210> 24 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> APOA2 leader sequence <400> 24 Met Lys Leu Leu Ala Ala Thr Val Leu Leu Leu Thr Ile Cys Ser Leu 1 5 10 15 Glu Gly          <210> 25 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> SEPP1 leader sequence <400> 25 Met Trp Arg Ser Leu Gly Leu Ala Leu Ala Leu Cys Leu Leu Pro Ser 1 5 10 15 Gly Gly Thr              <210> 26 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> VH1-02 leader sequence <400> 26 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Ala His Ser              <210> 27 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> VH1-46 leader sequence <400> 27 Met Asp Trp Thr Trp Arg Val Phe Cys Leu Leu Ala Val Ala Pro Gly 1 5 10 15 Ala His Ser              <210> 28 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> VH2-05 leader sequence <400> 28 Met Asp Thr Leu Cys Ser Thr Leu Leu Leu Leu Thr Ile Pro Ser Trp 1 5 10 15 Val Leu Ser              <210> 29 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> VH3-11 leader sequence <400> 29 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Ile Lys Gly 1 5 10 15 Val Gln Cys              <210> 30 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> VH4-04 leader sequence <400> 30 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser              <210> 31 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> VH5-51 leader sequence <400> 31 Met Gly Ser Thr Ala Ile Leu Ala Leu Leu Leu Ala Val Leu Gln Gly 1 5 10 15 Val Cys Ser              <210> 32 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> VH7-04 leader sequence <400> 32 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Ala His Ser              <210> 33 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> APOH leader sequence <400> 33 Met Ile Ser Pro Val Leu Ile Leu Phe Ser Ser Phe Leu Cys His Val 1 5 10 15 Ala Ile Ala              <210> 34 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> AMBP leader sequence <400> 34 Met Arg Ser Leu Gly Ala Leu Leu Leu Leu Leu Ser Ala Cys Leu Ala 1 5 10 15 Val Ser Ala              <210> 35 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> SERPIND1 leader sequence <400> 35 Met Lys His Ser Leu Asn Ale Leu Leu Ile Phe Leu Ile Ile Thr Ser 1 5 10 15 Ala Trp Gly              <210> 36 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> FGA leader sequence <400> 36 Met Phe Ser Met Arg Ile Val Cys Leu Val Leu Ser Val Val Gly Thr 1 5 10 15 Ala Trp Thr              <210> 37 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> F12 leader sequence <400> 37 Met Arg Ala Leu Leu Leu Leu Gly Phe Leu Leu Val Ser Leu Glu Ser 1 5 10 15 Thr Leu Ser              <210> 38 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> DEFA6 leader sequence <400> 38 Met Arg Thr Leu Thr Ile Leu Thr Ala Val Leu Leu Val Ala Leu Gln 1 5 10 15 Ala Lys Ala              <210> 39 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> LPA /// PLG leader sequence <400> 39 Met Glu His Lys Glu Val Val Leu Leu Leu Leu Leu Phe Leu Lys Ser 1 5 10 15 Ala Ala Pro              <210> 40 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> TF leader sequence <400> 40 Met Arg Leu Ala Val Gly Ala Leu Leu Val Cys Ala Val Leu Gly Leu 1 5 10 15 Cys Leu Ala              <210> 41 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> VH6-01 leader sequence <400> 41 Met Ser Val Ser Phe Leu Ile Phe Leu Pro Val Leu Gly Leu Pro Trp 1 5 10 15 Gly Val Leu Ser             20 <210> 42 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> VK-O11 leader sequence <400> 42 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Pro 1 5 10 15 Gly Ser Ser Glu             20 <210> 43 <211> 20 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > VK-L2 leader sequence <400> 43 Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly             20 <210> 44 <211> 20 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > VK-B3 leader sequence <400> 44 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly             20 <210> 45 <211> 20 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > VK-B2 leader sequence <400> 45 Met Gly Ser Gln Val His Leu Leu Ser Phe Leu Leu Leu Trp Ile Ser 1 5 10 15 Asp Thr Arg Ala             20 <210> 46 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> GPX3 leader sequence <400> 46 Met Ala Arg Leu Leu Gln Ala Ser Cys Leu Leu Ser Leu Leu Leu Ala 1 5 10 15 Gly Phe Val Ser             20 <210> 47 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> APOC3 leader sequence <400> 47 Met Gln Pro Arg Val Leu Le Val Val Ala Leu Leu Ala Leu Leu Ala 1 5 10 15 Ser Ala Arg Ala             20 <210> 48 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> APOA4 leader sequence <400> 48 Met Phe Leu Lys Ala Val Val Leu Thr Leu Ala Leu Val Ala Val Ala 1 5 10 15 Gly Ala Arg Ala             20 <210> 49 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> TTR leader sequence <400> 49 Met Ala Ser His Arg Leu Leu Leu Leu Cys Leu Ala Gly Leu Val Phe 1 5 10 15 Val Ser Glu Ala             20 <210> 50 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> IGFBP5 leader sequence <400> 50 Met Val Leu Leu Thu Ala Val Leu Leu Leu Leu Ala Ala Tyr Ala Gly 1 5 10 15 Pro Ala Gln Ser             20 <210> 51 <211> 20 <212> PRT <213> Artificial Sequence <220> <B23> B2M leader sequence <400> 51 Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser 1 5 10 15 Gly Leu Glu Ala             20 <210> 52 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> IGFBP4 leader sequence <400> 52 Met Leu Pro Leu Cys Leu Val Ala Leu Leu Leu Ala Ala Gly Pro 1 5 10 15 Gly Pro Ser Leu Gly             20 <210> 53 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> TFF3 leader sequence <400> 53 Met Ala Ala Arg Ala Leu Cys Met Leu Gly Leu Val Leu Ala Leu Leu 1 5 10 15 Ser Ser Ser Ala             20 <210> 54 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> GUCA2A leader sequence <400> 54 Met Asn Ala Phe Leu Leo Phe Ala Leu Cys Leu Leu Gly Ala Trp Ala 1 5 10 15 Ala Leu Ala Gly Gly             20 <210> 55 <211> 22 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > VK-A30 leader sequence <400> 55 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ala Arg Cys             20 <210> 56 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> APOC2 leader sequence <400> 56 Met Gly Thr Arg Leu Leu Pro Ala Leu Phe Leu Val Leu Leu Val Leu 1 5 10 15 Gly Phe Glu Val Gln Gly             20 <210> 57 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> CLU leader sequence <400> 57 Met Met Lys Thr Leu Leu Leu Phe Val Gly Leu Leu Leu Thr Trp Glu 1 5 10 15 Ser Gly Gln Val Leu Gly             20 <210> 58 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> PTGDS leader sequence <400> 58 Met Ala Thr His His Thr Leu Trp Met Gly Leu Ala Leu Leu Gly Val 1 5 10 15 Leu Gly Asp Leu Gln Ala             20 <210> 59 <211> 22 <212> PRT <213> Artificial Sequence <220> C3 leader sequence <400> 59 Met Gly Pro Thr Ser Gly Pro Ser Leu Leu Leu Leu Leu Leu Thr His 1 5 10 15 Leu Pro Leu Ala Leu Gly             20 <210> 60 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> F9 leader sequence <400> 60 Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr Ile Cys Leu Leu Gly 1 5 10 15 Tyr Leu Leu Ser Ala Glu Cys             20 <210> 61 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> A2M leader sequence <400> 61 Met Gly Lys Asn Lys Leu Leu His Pro Ser Leu Leu Leu Leu Leu 1 5 10 15 Val Leu Leu Pro Thr Asp Ala             20 <210> 62 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> SERPINA3 leader sequence <400> 62 Met Glu Arg Met Leu Pro Leu Leu Ala Leu Gly Leu Leu Ala Ala Gly 1 5 10 15 Phe Cys Pro Ala Val Leu Cys             20 <210> 63 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> SERPINA1 leader sequence <400> 63 Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala Gly Leu Cys 1 5 10 15 Cys Leu Val             20 <210> 64 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> AGT leader sequence <400> 64 Met Ala Pro Ala Gly Val Ser Leu Arg Ala Thr Ile Leu Cys Leu Leu 1 5 10 15 Ala Trp Ala Gly Leu Ala Ala Gly             20 <210> 65 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> BF leader sequence <400> 65 Met Gly Ser Asn Leu Ser Pro Gln Leu Cys Leu Met Pro Phe Ile Leu 1 5 10 15 Gly Leu Leu Ser Gly Gly Val Thr Thr             20 25 <210> 66 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> AGFBP7 leader sequence <400> 66 Met Glu Arg Pro Ser Leu Arg Ala Leu Leu Leu Gly Ala Ala Gly Leu 1 5 10 15 Leu Leu Leu Leu             20 25 <210> 67 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> APOC1 leader sequence <400> 67 Met Arg Leu Phe Leu Ser Leu Pro Val Leu Val Val Leu Ser Ile 1 5 10 15 Val Leu Glu Gly Pro Ala Pro Ala Gln Gly             20 25 <210> 68 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> GSN leader sequence <400> 68 Met Ala Pro His Arg Pro Ala Pro Ala Leu Leu Cys Ala Leu Ser Leu 1 5 10 15 Ala Leu Cys Ala Leu             20 25 <210> 69 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> APOB leader sequence <400> 69 Met Asp Pro Pro Arg Ala Leu Ala Leu Leu Ala Leu Pro Ala 1 5 10 15 Leu Leu Leu Leu Leu Leu             20 25 <210> 70 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> APOB leader sequence <400> 70 Met Gln Arg Ala Arg Pro Thr Leu Trp Ala Ala Leu Thr Leu Leu 1 5 10 15 Val Leu Leu Arg Gly Pro Pro Val Ala Arg Ala             20 25 <210> 71 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> ITIH1 leader sequence <400> 71 Met Asp Gly Ala Met Gly Pro Arg Gly Leu Leu Leu Cys Met Tyr Leu 1 5 10 15 Val Ser Leu Leu Ile Leu Gln             20 25 <210> 72 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> FGB leader sequence <400> 72 Met Val Ser Trp Ser Phe His Lys Leu Lys Thr Met Lys His Leu Leu 1 5 10 15 Leu Leu Leu Leu Cys Val Phe Leu Val Lys Ser             20 25 <210> 73 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> ITIH4 leader sequence <400> 73 Met Lys Pro Pro Arg Pro Val Arg Thr Cys Ser Lys Val Leu Val Leu 1 5 10 15 Leu Ser Leu Leu Ala Ile His Gln Thr Thr Thr Ala             20 25 <210> 74 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> RNASE1 leader sequence <400> 74 Met Ala Leu Glu Lys Ser Leu Val Arg Leu Leu Leu Leu Val Leu Ile 1 5 10 15 Leu Leu Val Leu Gly Trp Val Gln Pro Ser Leu Gly             20 25 <210> 75 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> FN1 leader sequence <400> 75 Met Leu Arg Gly Pro Gly Pro Gly Leu Leu Leu Leu Ala Val Gln Cys 1 5 10 15 Leu Gly Thr Ala Val Ser Thr Gly Ala Ser Lys Ser Lys Arg             20 25 30 <210> 76 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding IGHV leader sequence <400> 76 atgggatgga gctatatcat cctctttttg gtagcaacag ctacagatgt ccactcc 57 <210> 77 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding INS leader sequence <400> 77 atgcgcctcc tgcccctgct ggcgctgctg gccctctggg gacctgaccc agccgcagcc 60 <210> 78 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding PRSS1 leader sequence <400> 78 atgaatctac ttctgatcct tacctttgtt gcagctgctg ttgct 45 <210> 79 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding ELA3B leader sequence <400> 79 atgatgctcc ggctgctcag ttccctcctc cttgtggccg ttgcc 45 <210> 80 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding C1S leader sequence <400> 80 atgtggtgca ttgtcctgtt ttcacttttg gcatgggttt atgct 45 <210> 81 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding ELA2B leader sequence <400> 81 atgattagga ccctgctgct gtccactttg gtggctggag ccctcagt 48 <210> 82 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding P4HB leader sequence <400> 82 atgctgcgcc gcgctctgct gtgcctggcc gtggccgccc tggtgcgcgc c 51 <210> 83 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding ITIH3 leader sequence <400> 83 atgtgggtcc cggttgtctt cctcaccctg tccgtgacgt ggattggtgc t 51 <210> 84 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding ITIH3 leader sequence <400> 84 atggcatttg catggtggcc ctgtctcatc ttggctctgc tctccagctt ggcagcctct 60 <210> 85 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding CLPS leader sequence <400> 85 atggagaaga tcctgatcct cctgcttgtc gccctctctg tggcctatgc a 51 <210> 86 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding PNLIPRP1 leader sequence <400> 86 atgctgatct tctggacaat cacacttttc ctgctgggag cagccaaagg a 51 <210> 87 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding AZGP1 leader sequence <400> 87 atggtgcctg tcctgctgtc tctgctgctg cttctgggtc ctgctgtccc c 51 <210> 88 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding PNLIPRP2 leader sequence <400> 88 atgctgcccc cttggaccct cggccttctc ctgctggcca cagtcagagg a 51 <210> 89 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding C1R leader sequence <400> 89 atgtggctct tgtacctcct ggtgccggcc ctgttctgca gggcaggagg c 51 <210> 90 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding SERPINF1 leader sequence   <400> 90 atgcaggccc tggtgctact cctctgcatt ggagccctcc tcgggcacag cagc 54 <210> 91 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding APOE leader sequence <400> 91 atgaaggttc tgtgggctgc gttgctggtc acattcctgg caggatgcca ggcc 54 <210> 92 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding APOA1 leader sequence <400> 92 atgaaagctg cggtgctgac cttggccgtg ctcttcctga cggggagcca ggct 54 <210> 93 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding ITIH2 leader sequence <400> 93 atgaaaagac tcacgtgctt tttcatctgc ttctttcttt ctgaagtatc aggc 54 <210> 94 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding CTRB1 leader sequence <400> 94 atggctttcc tctggctcct ctcctgctgg gccctcctgg gtaccacctt cggc 54 <210> 95 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding KNG1 leader sequence <400> 95 atgaaactaa ttaccatcct tttcctctgc tccaggctgc tactaagttt aacc 54 <210> 96 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding HP leader sequence <400> 96 atgagtgccc tgggagctgt cattgccctc ctgctctggg gacagctttt tgca 54 <210> 97 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding SAA4 leader sequence <400> 97 atgaggcttt tcacaggcat tgttttctgc tccttggtca tgggagtcac cagt 54 <210> 98 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding SAA1 leader sequence <400> 98 atgaagcttc tcacgggcct ggttttctgc tccttggtcc tgggtgtcag cagc 54 <210> 99 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding APOA2 leader sequence <400> 99 atgaagctgc tcgcagcaac tgtgctactc ctcaccatct gcagccttga agga 54 <210> 100 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding SEPP1 leader sequence <400> 100 atgtggagaa gcctggggct tgccctggct ctctgtctcc tcccatcggg aggaaca 57 <210> 101 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding VH1-02 leader sequence <400> 101 atggactgga cctggaggat cctcttcttg gtggcagcag ccacaggagc ccactcc 57 <210> 102 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding VH1-46 leader sequence <400> 102 atggactgga cctggagggt cttctgcttg ctggctgtag ctccaggagc tcactcc 57 <210> 103 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding VH2-05 leader sequence <400> 103 atggacacac tttgctccac gctcctgctg ctgaccatcc cttcatgggt cttgtcc 57 <210> 104 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding VH3-11 leader sequence <400> 104 atggaatttg ggctgagctg ggttttcctt gttgctatta taaaaggtgt ccagtgt 57 <210> 105 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding VH4-04 leader sequence <400> 105 atggaatttg ggctgagctg ggttttcctt gttgctatta taaaaggtgt ccagtgt 57 <210> 106 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding VH5-51 leader sequence <400> 106 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgttcc 57 <210> 107 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding VH7-04 leader sequence <400> 107 atggactgga cctggaggat cctcttcttg gtggcagcag caacaggtgc ccactcc 57 <210> 108 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding APOH leader sequence <400> 108 atgatttctc cagtgctcat cttgttctcg agttttctct gccatgttgc tattgca 57 <210> 109 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding AMBP leader sequence <400> 109 atgaggagcc tcggggccct gctcttgctg ctgagcgcct gcctggcggt gagcgct 57 <210> 110 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding SERPIND1 leader sequence <400> 110 atgaaacact cattaaacgc acttctcatt ttcctcatca taacatctgc gtggggt 57 <210> 111 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding FGA leader sequence <400> 111 atgttttcca tgaggatcgt ctgcctggtc ctaagtgtgg tgggcacagc atggact 57 <210> 112 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding F12 leader sequence <400> 112 atgagggctc tgctgctcct ggggttcctg ctggtgagct tggagtcaac actttcg 57 <210> 113 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding DEFA6 leader sequence <400> 113 atgagaaccc tcaccatcct cactgctgtt ctcctcgtgg ccctccaggc caaggct 57 <210> 114 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding LPA /// PLG leader sequence <400> 114 atggaacata aggaagtggt tcttctactt cttttatttc tgaaatcagc agcacct 57 <210> 115 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding TF leader sequence <400> 115 atgaggctcg ccgtgggagc cctgctggtc tgcgccgtcc tggggctgtg tctggct 57 <210> 116 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding VH6-01 leader sequence <400> 116 atgtctgtct ccttcctcat cttcctgccc gtgctgggcc tcccatgggg tgtcctgtca 60 <210> 117 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding VK-O11 leader sequence <400> 117 atgaggctcc ctgctcagct cctggggctg ctaatgctct gggtccctgg atccagtgag 60 <210> 118 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding VK-L2 leader sequence <400> 118 atggaagccc cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60 <210> 119 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding VK-B3 leader sequence <400> 119 atggtgttgc agacccaggt cttcatttct ctgttgctct ggatctctgg tgcctacggg 60 <210> 120 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding VK-B2 leader sequence <400> 120 atggggtccc aggttcacct cctcagcttc ctcctccttt ggatctctga taccagggca 60 <210> 121 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding GPX3 leader sequence <400> 121 atggcccggc tgctgcaggc gtcctgcctg ctttccctgc tcctggccgg cttcgtctcg 60 <210> 122 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding APOC3 leader sequence <400> 122 atgcagcccc gggtactcct tgttgttgcc ctcctggcgc tcctggcctc tgcccgagct 60 <210> 123 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding APOA4 leader sequence <400> 123 atgttcctga aggccgtggt cctgaccctg gccctggtgg ctgtcgccgg agccagggct 60 <210> 124 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding TTR leader sequence <400> 124 atggcttctc atcgtctgct cctcctctgc cttgctggac tggtatttgt gtctgaggct 60 <210> 125 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding IGFBP5 leader sequence <400> 125 atggtgttgc tcaccgcggt cctcctgctg ctggccgcct atgcggggcc ggcccagagc 60 <210> 126 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding B2M leader sequence <400> 126 atgtctcgct ccgtggcctt agctgtgctc gcgctactct ctctttctgg cctggaggct 60 <210> 127 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding IGFBP4 leader sequence <400> 127 atgctgcccc tctgcctcgt ggccgccctg ctgctggccg ccgggcccgg gccgagcctg 60 ggc 63 <210> 128 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding TFF3 leader sequence <400> 128 atggctgcca gagcgctctg catgctgggg ctggtcctgg ccttgctgtc ctccagctct 60 gct 63 <210> 129 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding GUCA2A leader sequence <400> 129 atgaatgcct tcctgctctt cgcactgtgc ctccttgggg cctgggccgc cttggcagga 60 ggg 63 <210> 130 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding VK-A30 leader sequence <400> 130 atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggtt cccaggtgcc 60 agatgt 66 <210> 131 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding APOC2 leader sequence <400> 131 atgggcacac gactcctccc agctctgttt cttgtcctcc tggtattggg atttgaggtc 60 cagggg 66 <210> 132 <211> 66 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > polynucleotide encoding CLU leader sequence <400> 132 atgatgaaga ctctgctgct gtttgtgggg ctgctgctga cctgggagag tgggcaggtc 60 ctgggg 66 <210> 133 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding PTGDS leader sequence <400> 133 atggctactc atcacacgct gtggatggga ctggccctgc tgggggtgct gggcgacctg 60 caggca 66 <210> 134 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding C3 leader sequence <400> 134 atgggaccca cctcaggtcc cagcctgctg ctcctgctac taacccacct ccccctggct 60 ctgggg 66 <210> 135 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding F9 leader sequence <400> 135 atgatcatgg cagaatcacc aggcctcatc accatctgcc ttttaggata tctactcagt 60 gctgaatgt 69 <210> 136 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding A2M leader sequence <400> 136 atggggaaga acaaactcct tcatccaagt ctggttcttc tcctcttggt cctcctgccc 60 acagacgcc 69 <210> 137 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding SERPINA3 leader sequence <400> 137 atggagagaa tgttacctct cctggctctg gggctcttgg cggctgggtt ctgccctgct 60 gtcctctgc 69 <210> 138 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding SERPINA1 leader sequence <400> 138 atgccgtctt ctgtctcgtg gggcatcctc ctgctggcag gcctgtgctg cctggtccct 60 gtctccctg 69 <210> 139 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding AGT leader sequence <400> 139 atggctcctg ccggtgtgag cctgagggcc accatcctct gcctcctggc ctgggctggc 60 ctggctgcag gt 72 <210> 140 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding BF leader sequence <400> 140 atggggagca atctcagccc ccaactctgc ctgatgccct ttatcttggg cctcttgtct 60 ggaggtgtga ccacc 75 <210> 141 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding IGFBP7 leader sequence <400> 141 atggagcggc cgtcgctgcg cgccctgctc ctcggcgccg ctgggctgct gctcctgctc 60 ctgcccctct cctcttcc 78 <210> 142 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding APOC1 leader sequence <400> 142 atgaggctct tcctgtcgct cccggtcctg gtggtggttc tgtcgatcgt cttggaaggc 60 ccagccccag cccagggg 78 <210> 143 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding GSN leader sequence <400> 143 atggctccgc accgccccgc gcccgcgctg ctttgcgcgc tgtccctggc gctgtgcgcg 60 ctgtcgctgc ccgtccgcgc g 81 <210> 144 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding APOB leader sequence <400> 144 atggacccgc cgaggcccgc gctgctggcg ctgctggcgc tgcctgcgct gctgctgctg 60 ctgctggcgg gcgccagg 78 <210> 145 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding IGFBP3 leader sequence <400> 145 atgcagcggg cgcgacccac gctctgggcc gctgcgctga ctctgctggt gctgctccgc 60 gggccgccgg tggcgcgggc t 81 <210> 146 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding ITIH1 leader sequence <400> 146 atggacggtg ccatggggcc tcgggggctg ctgttgtgca tgtacctggt atctctcctc 60 atcctgcagg ccatgcctgc c 81 <210> 147 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding FGB leader sequence <400> 147 atggtttctt ggagcttcca caaacttaaa accatgaaac atctattatt gctactattg 60 tgtgtttttc tagttaagtc c 81 <210> 148 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding ITIH4 leader sequence <400> 148 atgaagcccc caaggcctgt ccgtacctgc agcaaagttc tcgtcctgct ttcactgctg 60 gccatccacc agactactac tgcc 84 <210> 149 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding RNASE1 leader sequence <400> 149 atggctctgg agaagtctct tgtccggctc cttctgcttg tcctgatact gctggtgctg 60 ggctgggtcc agccttccct gggc 84 <210> 150 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding FN1 leader sequence <400> 150 atgcttaggg gtccggggcc cgggctgctg ctgctggccg tccagtgcct ggggacagcg 60 gtgccctcca cgggagcctc gaagagcaag agg 93 <210> 151 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for oriP <400> 151 gtagatctgc aggaaaagga caagc 25 <210> 152 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for oriP <400> 152 cgagatctgg ttgacttccc taatgt 26 <210> 153 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Fc gamma <400> 153 cgggatccgg ccgtgggggc cgacaaaact cacacatgcc 40 <210> 154 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Fc gamma <400> 154 cgagtctcat ttacccggag acaggga 27 <210> 155 <211> 280 <212> PRT <213> Artificial Sequence <220> <223> soluble human CD1D <400> 155 Val Pro Gln Arg Leu Phe Pro Leu Arg Cys Leu Gln Ile Ser Ser Phe 1 5 10 15 Ala Asn Ser Ser Trp Thr Arg Thr Asp Gly Leu Ala Trp Leu Gly Glu             20 25 30 Leu Gln Thr His Ser Trp Ser Asn Asp Ser Asp Thr Val Arg Ser Leu         35 40 45 Lys Pro Trp Ser Gln Gly Thr Phe Ser Asp Gln Gln Trp Glu Thr Leu     50 55 60 Gln His Ile Phe Arg Val Tyr Arg Ser Ser Phe Thr Arg Asp Val Lys 65 70 75 80 Glu Phe Ala Lys Met Leu Arg Leu Ser Tyr Pro Leu Glu Leu Gln Val                 85 90 95 Ser Ala Gly Cys Glu Val His Pro Gly Asn Ala Ser Asn Asn Phe Phe             100 105 110 His Val Ala Phe Gln Gly Lys Asp Ile Leu Ser Phe Gln Gly Thr Ser         115 120 125 Trp Glu Pro Thr Gln Glu Ala Pro Leu Trp Val Asn Leu Ala Ile Gln     130 135 140 Val Leu Asn Gln Asp Lys Trp Thr Arg Glu Thr Val Gln Trp Leu Leu 145 150 155 160 Asn Gly Thr Cys Pro Gln Phe Val Ser Gly Leu Leu Glu Ser Gly Lys                 165 170 175 Ser Glu Leu Lys Lys Gln Val Lys Pro Lys Ala Trp Leu Ser Arg Gly             180 185 190 Pro Ser Pro Gly Pro Gly Arg Leu Leu Leu Val Cys His Val Ser Gly         195 200 205 Phe Tyr Pro Lys Pro Val Trp Val Lys Trp Met Arg Gly Glu Gln Glu     210 215 220 Gln Gln Gly Thr Gln Pro Gly Asp Ile Leu Pro Asn Ala Asp Glu Thr 225 230 235 240 Trp Tyr Leu Arg Ala Thr Leu Asp Val Val Ala Gly Glu Ala Ala Gly                 245 250 255 Leu Ser Cys Arg Val Lys His Ser Ser Leu Glu Gly Gln Asp Ile Val             260 265 270 Leu Tyr Trp Gly Gly Ser Tyr Thr         275 280 <210> 156 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> forward primer for soluble human CD1D <400> 156 cagggggccg tgggggccgt cccgcaaagg cttttccc 38 <210> 157 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer ofr soluble human CD1D <400> 157 tagcggccgt gggggccgga ggtgtagctc ccacc 35 <210> 158 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> S00 forward primer <400> 158 gggctagcgg tcaccgccac catgggatgg agctatatca tcctcttttt ggtagcaaca 60 gctacagatg tccactccca gggggccgtg ggg 93 <210> 159 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> S01 forward primer <400> 159 ctagcggtca ccgccaccat gcgcctcctg cccctgctgg cgctgctggc cctctgggga 60 cctgacccag ccgcagccgg ggccgtgggg g 91 <210> 160 <211> 79 <212> DNA <213> Artificial Sequence <220> <223> S02 forward primer <400> 160 ctagcggtca ccgccaccat gaatctactt ctgatcctta cctttgttgc agctgctgtt 60 gctgctgggg ccgtggggg 79 <210> 161 <211> 79 <212> DNA <213> Artificial Sequence <220> <223> S03 forward primer <400> 161 ctagcggtca ccgccaccat gatgctccgg ctgctcagtt ccctcctcct tgtggccgtt 60 gcctctgggg ccgtggggg 79 <210> 162 <211> 79 <212> DNA <213> Artificial Sequence <220> <223> S04 forward primer <400> 162 ctagcggtca ccgccaccat gtggtgcatt gtcctgtttt cacttttggc atgggtttat 60 gctgaagggg ccgtggggg 79 <210> 163 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> S05 forward primer <400> 163 ctagcggtca ccgccaccat gattaggacc ctgctgctgt ccactttggt ggctggagcc 60 ctcagtgaag gggccgtggg gg 82 <210> 164 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> S06 forward primer <400> 164 ctagcggtca ccgccaccat gctgcgccgc gctctgctgt gcctggccgt ggccgccctg 60 gtgcgcgccg acggggccgt ggggg 85 <210> 165 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> S07 forward primer <400> 165 ctagcggtca ccgccaccat gtgggtcccg gttgtcttcc tcaccctgtc cgtgacgtgg 60 attggtgctg ctggggccgt ggggg 85 <210> 166 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> S08 forward primer <400> 166 ctagcggtca ccgccaccat ggcatttgca tggtggccct gtctcatctt ggctctgctc 60 tccagcttgg cagcctctgg ggccgtgggg g 91 <210> 167 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> S09 forward primer <400> 167 ctagcggtca ccgccaccat ggagaagatc ctgatcctcc tgcttgtcgc cctctctgtg 60 gcctatgcag caggggccgt ggggg 85 <210> 168 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> S10 forward primer <400> 168 ctagcggtca ccgccaccat gctgatcttc tggacaatca cacttttcct gctgggagca 60 gccaaaggaa aaggggccgt ggggg 85 <210> 169 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> S11 forward primer <400> 169 ctagcggtca ccgccaccat ggtgcctgtc ctgctgtctc tgctgctgct tctgggtcct 60 gctgtccccc agggggccgt ggggg 85 <210> 170 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> S12 forward primer <400> 170 ctagcggtca ccgccaccat gctgccccct tggaccctcg gccttctcct gctggccaca 60 gtcagaggaa aaggggccgt ggggg 85 <210> 171 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> S13 forward primer <400> 171 ctagcggtca ccgccaccat gtggctcttg tacctcctgg tgccggccct gttctgcagg 60 gcaggaggct ctggggccgt ggggg 85 <210> 172 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> S14 forward primer <400> 172 ctagcggtca ccgccaccat gcaggccctg gtgctactcc tctgcattgg agccctcctc 60 gggcacagca gccagggggc cgtggggg 88 <210> 173 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> S15 forward primer <400> 173 ctagcggtca ccgccaccat gaaggttctg tgggctgcgt tgctggtcac attcctggca 60 ggatgccagg ccaaaggggc cgtggggg 88 <210> 174 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> S16 forward primer <400> 174 ctagcggtca ccgccaccat gaaagctgcg gtgctgacct tggccgtgct cttcctgacg 60 gggagccagg ctcgaggggc cgtggggg 88 <210> 175 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> S17 forward primer <400> 175 ctagcggtca ccgccaccat gaaaagactc acgtgctttt tcatctgctt ctttctttct 60 gaagtatcag gcttcggggc cgtggggg 88 <210> 176 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> S18 forward primer <400> 176 ctagcggtca ccgccaccat ggctttcctc tggctcctct cctgctgggc cctcctgggt 60 accaccttcg gcaaaggggc cgtggggg 88 <210> 177 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> S19 forward primer <400> 177 ctagcggtca ccgccaccat gaaactaatt accatccttt tcctctgctc caggctgcta 60 ctaagtttaa cccagggggc cgtggggg 88 <210> 178 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> S20 forward primer <400> 178 ctagcggtca ccgccaccat gagtgccctg ggagctgtca ttgccctcct gctctgggga 60 cagctttttg cagtgggggc cgtggggg 88 <210> 179 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> S21 forward primer <400> 179 ctagcggtca ccgccaccat gaggcttttc acaggcattg ttttctgctc cttggtcatg 60 ggagtcacca gtgaaggggc cgtggggg 88 <210> 180 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> S22 forward primer <400> 180 ctagcggtca ccgccaccat gaagcttctc acgggcctgg ttttctgctc cttggtcctg 60 ggtgtcagca gccgaggggc cgtggggg 88 <210> 181 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> S23 forward primer <400> 181 ctagcggtca ccgccaccat gaagctgctc gcagcaactg tgctactcct caccatctgc 60 agccttgaag gagcaggggc cgtggggg 88 <210> 182 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> S24 forward primer <400> 182 ctagcggtca ccgccaccat gtggagaagc ctggggcttg ccctggctct ctgtctcctc 60 ccatcgggag gaacagaagg ggccgtgggg g 91 <210> 183 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> S25 forward primer <400> 183 ctagcggtca ccgccaccat ggactggacc tggaggatcc tcttcttggt ggcagcagcc 60 acaggagccc actcccaggg ggccgtgggg g 91 <210> 184 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> S26 forward primer <400> 184 ctagcggtca ccgccaccat ggactggacc tggagggtct tctgcttgct ggctgtagct 60 ccaggagctc actcccaggg ggccgtgggg g 91 <210> 185 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> S27 forward primer <400> 185 ctagcggtca ccgccaccat ggacacactt tgctccacgc tcctgctgct gaccatccct 60 tcatgggtct tgtcccaggg ggccgtgggg g 91 <210> 186 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> S28 forward primer <400> 186 ctagcggtca ccgccaccat ggaatttggg ctgagctggg ttttccttgt tgctattata 60 aaaggtgtcc agtgtcaggg ggccgtgggg g 91 <210> 187 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> S29 forward primer <400> 187 ctagcggtca ccgccaccat ggaacacctg tggttcttcc tcctgctggt ggcagctccc 60 agatgggtcc tgtcccaggg ggccgtgggg g 91 <210> 188 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> S30 forward primer <400> 188 ctagcggtca ccgccaccat ggggtcaacc gccatcctcg ccctcctcct ggctgttctc 60 caaggagtct gttccgaggg ggccgtgggg g 91 <210> 189 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> S31 forward primer <400> 189 ctagcggtca ccgccaccat ggactggacc tggaggatcc tcttcttggt ggcagcagca 60 acaggtgccc actcccaggg ggccgtgggg g 91 <210> 190 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> S32 forward primer <400> 190 ctagcggtca ccgccaccat gatttctcca gtgctcatct tgttctcgag ttttctctgc 60 catgttgcta ttgcaggggc cgtggggg 88 <210> 191 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> S33 forward primer <400> 191 ctagcggtca ccgccaccat gaggagcctc ggggccctgc tcttgctgct gagcgcctgc 60 ctggcggtga gcgctggggc cgtggggg 88 <210> 192 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> S34 forward primer <400> 192 ctagcggtca ccgccaccat gaaacactca ttaaacgcac ttctcatttt cctcatcata 60 acatctgcgt ggggtggggc cgtggggg 88 <210> 193 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> S35 forward primer <400> 193 ctagcggtca ccgccaccat gttttccatg aggatcgtct gcctggtcct aagtgtggtg 60 ggcacagcat ggactgcagg ggccgtgggg g 91 <210> 194 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> S36 forward primer <400> 194 ctagcggtca ccgccaccat gagggctctg ctgctcctgg ggttcctgct ggtgagcttg 60 gagtcaacac tttcgatcgg ggccgtgggg g 91 <210> 195 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> S37 forward primer <400> 195 ctagcggtca ccgccaccat gagaaccctc accatcctca ctgctgttct cctcgtggcc 60 ctccaggcca aggctgaagg ggccgtgggg g 91 <210> 196 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> S38 forward primer <400> 196 ctagcggtca ccgccaccat ggaacataag gaagtggttc ttctacttct tttatttctg 60 aaatcagcag cacctgaagg ggccgtgggg g 91 <210> 197 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> S39 forward primer <400> 197 ctagcggtca ccgccaccat gaggctcgcc gtgggagccc tgctggtctg cgccgtcctg 60 gggctgtgtc tggctgtggg ggccgtgggg g 91 <210> 198 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> S40 forward primer <400> 198 ctagcggtca ccgccaccat gtctgtctcc ttcctcatct tcctgcccgt gctgggcctc 60 ccatggggtg tcctgtcaca gggggccgtg gggg 94 <210> 199 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> S41 forward primer <400> 199 ctagcggtca ccgccaccat gaggctccct gctcagctcc tggggctgct aatgctctgg 60 gtccctggat ccagtgagga tggggccgtg gggg 94 <210> 200 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> S42 forward primer <400> 200 ctagcggtca ccgccaccat ggaagcccca gcgcagcttc tcttcctcct gctactctgg 60 ctcccagata ccaccggaga aggggccgtg gggg 94 <210> 201 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> S43 forward primer <400> 201 ctagcggtca ccgccaccat ggtgttgcag acccaggtct tcatttctct gttgctctgg 60 atctctggtg cctacgggga cggggccgtg gggg 94 <210> 202 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> S44 forward primer <400> 202 ctagcggtca ccgccaccat ggggtcccag gttcacctcc tcagcttcct cctcctttgg 60 atctctgata ccagggcaga aggggccgtg gggg 94 <210> 203 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> S45 forward primer <400> 203 ctagcggtca ccgccaccat ggcccggctg ctgcaggcgt cctgcctgct ttccctgctc 60 ctggccggct tcgtctcgca gggggccgtg gggg 94 <210> 204 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> S46 forward primer <400> 204 ctagcggtca ccgccaccat gcagccccgg gtactccttg ttgttgccct cctggcgctc 60 ctggcctctg cccgagcttc tggggccgtg gggg 94 <210> 205 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> S47 forward primer <400> 205 ctagcggtca ccgccaccat gttcctgaag gccgtggtcc tgaccctggc cctggtggct 60 gtcgccggag ccagggctga aggggccgtg gggg 94 <210> 206 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> S48 forward primer <400> 206 ctagcggtca ccgccaccat ggcttctcat cgtctgctcc tcctctgcct tgctggactg 60 gtatttgtgt ctgaggctgg ggccgtgggg g 91 <210> 207 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> S49 forward primer <400> 207 ctagcggtca ccgccaccat ggtgttgctc accgcggtcc tcctgctgct ggccgcctat 60 gcggggccgg cccagagcct gggggccgtg gggg 94 <210> 208 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> S5O forward primer <400> 208 ctagcggtca ccgccaccat gtctcgctcc gtggccttag ctgtgctcgc gctactctct 60 ctttctggcc tggaggctat cggggccgtg gggg 94 <210> 209 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S51A forward primer <400> 209 ctagcggtca ccgccaccat ggtgcccctc tgcctcgtgg ccgccctgct gctggccgcc 60 gggcc 65 <210> 210 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S52A forward primer <400> 210 ctagcggtca ccgccaccat ggctgccaga gcgctctgca tgctggggct ggtcctggcc 60 ttgct 65 <210> 211 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S53A forward primer <400> 211 ctagcggtca ccgccaccat gaatgccttc ctgctcttcg cactgtgcct ccttggggcc 60 tgggc 65 <210> 212 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S54A forward primer <400> 212 ctagcggtca ccgccaccat ggacatgagg gtccccgctc agctcctggg gctcctgcta 60 ctctg 65 <210> 213 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S55A forward primer <400> 213 ctagcggtca ccgccaccat gggcacacga ctcctcccag ctctgtttct tgtcctcctg 60 gtatt 65 <210> 214 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S56A forward primer <400> 214 ctagcggtca ccgccaccat gatgaagact ctgctgctgt ttgtggggct gctgctgacc 60 tggga 65 <210> 215 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S57A forward primer <400> 215 ctagcggtca ccgccaccat ggctactcat cacacgctgt ggatgggact ggccctgctg 60 ggggt 65 <210> 216 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S58A forward primer <400> 216 ctagcggtca ccgccaccat gggacccacc tcaggtccca gcctgctgct cctgctacta 60 accca 65 <210> 217 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S59A forward primer <400> 217 ctagcggtca ccgccaccat gatcatggca gaatcaccag gcctcatcac catctgcctt 60 TTagG 65 <210> 218 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S60A forward primer <400> 218 ctagcggtca ccgccaccat ggggaagaac aaactccttc atccaagtct ggttcttctc 60 ctctt 65 <210> 219 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S61A forward primer <400> 219 ctagcggtca ccgccaccat ggagagaatg ttacctctcc tggctctggg gctcttggcg 60 gctgg 65 <210> 220 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S62A forward primer <400> 220 ctagcggtca ccgccaccat gccgtcttct gtctcgtggg gcatcctcct gctggcaggc 60 ctgtg 65 <210> 221 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S63A forward primer <400> 221 ctagcggtca ccgccaccat ggctcctgcc ggtgtgagcc tgagggccac catcctctgc 60 ctcct 65 <210> 222 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S64A forward primer <400> 222 ctagcggtca ccgccaccat ggggagcaat ctcagccccc aactctgcct gatgcccttt 60 atctt 65 <210> 223 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S65A forward primer <400> 223 ctagcggtca ccgccaccat ggagcgcgcg tcgctgcgcg ccctgctctt cggccccgct 60 gggct 65 <210> 224 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S66A forward primer <400> 224 ctagcggtca ccgccaccat gaggctcttc ctgtcgctcc cggtcctggt ggtggttctg 60 tcgat 65 <210> 225 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S67A forward primer <400> 225 ctagcggtca ccgccaccat ggctccgcac cgccccgcgc ccgcgctgct ttgcgcgctg 60 tccct 65 <210> 226 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S68A forward primer <400> 226 ctagcggtca ccgccaccat ggacccgccg aggcccgcgc tgctggcgct gctggcgctg 60 cctgc 65 <210> 227 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S69A forward primer <400> 227 ctagcggtca ccgccaccat gcagcgggcg cgacccacgc tctgggccgc tgcgctgact 60 ctgct 65 <210> 228 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S70A forward primer <400> 228 ctagcggtca ccgccaccat ggacggtgcc atggggcctc gggggctgct gttgtgcatg 60 tacct 65 <210> 229 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S71A forward primer <400> 229 ctagcggtca ccgccaccat ggtttcttgg agcttccaca aacttaaaac catgaaacat 60 CTatT 65 <210> 230 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S72A forward primer <400> 230 ctagcggtca ccgccaccat gaagccccca aggcctgtcc gtacctgcag caaagttctc 60 gtcct 65 <210> 231 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S73A forward primer <400> 231 ctagcggtca ccgccaccat ggctctggag aagtctcttg tccggctcct tctgcttgtc 60 ctgat 65 <210> 232 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> S74A forward primer <400> 232 ctagcggtca ccgccaccat gcttaggggt ccggggcccg ggctgctgct gctggccgtc 60 cagtgcctgg ggac 74 <210> 233 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S51B forward primer <400> 233 gctgctggcc gccgggcccg ggccgagcct gggcgatggg gccgtggggg 50 <210> 234 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S52B forward primer <400> 234 gctggtcctg gccttgctgt cctccagctc tgctgaaggg gccgtggggg 50 <210> 235 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S53B forward primer <400> 235 cctccttggg gcctgggccg ccttggcagg aggggtgggg gccgtggggg 50 <210> 236 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> S54B forward primer <400> 236 ggggctcctg ctactctggt tcccaggtgc cagatgtgac ggggccgtgg ggg 53 <210> 237 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> S55B forward primer <400> 237 tcttgtcctc ctggtattgg gatttgaggt ccaggggacc ggggccgtgg ggg 53 <210> 238 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> S56B forward primer <400> 238 gctgctgctg acctgggaga gtgggcaggt cctgggggat ggggccgtgg ggg 53 <210> 239 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> S57B forward primer <400> 239 actggccctg ctgggggtgc tgggcgacct gcaggcagca ggggccgtgg ggg 53 <210> 240 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> S58B forward primer <400> 240 gctcctgcta ctaacccacc tccccctggc tctggggtct ggggccgtgg ggg 53 <210> 241 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> S59B forward primer <400> 241 caccatctgc cttttaggat atctactcag tgctgaatgt actggggccg tggggg 56 <210> 242 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> S60B forward primer <400> 242 tctggttctt ctcctcttgg tcctcctgcc cacagacgcc tctggggccg tggggg 56 <210> 243 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> S61B forward primer <400> 243 ggggctcttg gcggctgggt tctgccctgc tgtcctctgc cacggggccg tggggg 56 <210> 244 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> S62B forward primer <400> 244 cctgctggca ggcctgtgct gcctggtccc tgtctccctg gctgaagggg ccgtggggg 59 <210> 245 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> S63B forward primer <400> 245 caccatcctc tgcctcctgg cctgggctgg cctggctgca ggtgatgggg ccgtggggg 59 <210> 246 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> S64B forward primer <400> 246 cctgatgccc tttatcttgg gcctcttgtc tggaggtgtg accaccactg gggccgtggg 60 gg 62 <210> 247 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S65B forward primer <400> 247 cttcggcccc gctgggctgc tgctcctgct cctgcccctc tcctcttcct ctggggccgt 60 ggggg 65 <210> 248 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S66B forward primer <400> 248 ggtggtggtt ctgtcgatcg tcttggaagg cccagcccca gcccagggga ctggggccgt 60 ggggg 65 <210> 249 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> S67B forward primer <400> 249 gctttgcgcg ctgtccctgg cgctgtgcgc gctgtcgctg cccgtccgcg cggcaggggc 60 cgtggggg 68 <210> 250 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> S68B forward primer <400> 250 gctgctggcg ctgcctgcgc tgctgctgct gctgctggcg ggcgccaggg ccgaaggggc 60 cgtggggg 68 <210> 251 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> S69B forward primer <400> 251 cgctgcgctg actctgctgg tgctgctccg cgggccgccg gtggcgcggg ctggggccgt 60 ggggg 65 <210> 252 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> S70B forward primer <400> 252 gctgttgtgc atgtacctgg tatctctcct catcctgcag gccatgcctg ccctgggggc 60 cgtggggg 68 <210> 253 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> S71B forward primer <400> 253 aaccatgaaa catctattat tgctactatt gtgtgttttt ctagttaagt cccagggggc 60 cgtggggg 68 <210> 254 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> S72B forward primer <400> 254 cagcaaagtt ctcgtcctgc tttcactgct ggccatccac cagaccacta ctgccgaagg 60 ggccgtgggg g 71 <210> 255 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> S73B forward primer <400> 255 ccttctgctt gtcctgatac tgctggtgct gggctgggtc cagccttccc tgggcaaagg 60 ggccgtgggg g 71 <210> 256 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> S74B forward primer <400> 256 cgtccagtgc ctggggacag cggtgccctc cacgggagcc tcgaagagca agaggcaggg 60 ggccgtgggg g 71 <210> 257 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 257 ttggcgaatt ccttcacgtc cctggtgaag ctgcttcggg taacccgaa 49

Claims (30)

A polynucleotide encoding a leader sequence peptide of SEQ ID NO: 8, and a polynucleotide encoding a human antibody Fc fragment operatively linked to a promoter of the framework vector. delete The recombinant vector according to claim 1, wherein the polynucleotide encoding the leader sequence peptide is a nucleic acid sequence of SEQ ID NO: 83. delete delete 2. The recombinant vector according to claim 1, wherein the skeletal vector is a mammalian expression vector. 7. The recombinant vector according to claim 6, wherein the mammalian expression vector is selected from the group consisting of pcDNA, pCMV, pEF, pSI, pCI, pCI-neo, pTK-neo, pFLAG and p3xFLAG. 2. The recombinant vector according to claim 1, wherein the skeletal vector is a vector having an oriP replication origin inserted from Epstein-Barr virus. delete 2. The recombinant vector of claim 1, further comprising a polynucleotide encoding an exogenous protein operably linked to a polynucleotide encoding the leader sequence peptide. 11. The recombinant vector according to claim 10, wherein the foreign protein is a growth factor, a soluble receptor, a cytokine or a monoclonal antibody. 12. The method of claim 11, wherein the growth factor is selected from the group consisting of transforming growth factor (TGF-beta), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM- Neurotrophin, platelet derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF-8), growth differentiation factor-9 (GDF9) A growth factor (aFGF or FGF-1), a basic fibroblast growth factor (bFGF or FGF-2), an epithelial growth factor (EGF), a hepatocyte growth factor (HGF), an osteogenic factor (BMP) ), The soluble receptor is any one selected from the group consisting of a G-protein related receptor, a receptor tyrosine kinase, or an ionotropic receptor, or a combination thereof, Cain is an interleukin-2 (IL-2), interferon (INF) Kind-10, interleukin-1, interleukin-12, or chemokine, or a combination thereof. A transfected host cell prepared by transfecting a host cell with the recombinant vector of claim 1. 14. The method according to claim 13, wherein said host cell is HEK-293E cells, CHO (Chinese hamster ovary) cells, BHK (baby hamster kidney) cells, NIH-3T3 cells, HEK-293T cells or COS-7 cells Transfected host cells. i) operably inserting a polynucleotide encoding an exogenous protein downstream of a polynucleotide encoding said leader sequence peptide in a recombinant vector comprising a polynucleotide encoding a leader sequence peptide of SEQ ID NO: 8 to produce a recombinant expression vector ; Ii) transfecting the host cell with the recombinant expression vector to prepare a transfected host cell; Iii) culturing the transfected host cell; And Vi) recovering the exogenous protein from the culture supernatant. 16. The method of claim 15, wherein the exogenous protein is a growth factor, a soluble receptor, a cytokine, or a monoclonal antibody. 18. The method of claim 16, wherein the growth factor is selected from the group consisting of a transforming growth factor (TGF-beta), a granulocyte-colony stimulating factor (G-CSF), a granulocyte-macrophage colony stimulating factor (GM- Neurotrophin, platelet derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF-8), growth differentiation factor-9 (GDF9) A growth factor (aFGF or FGF-1), a basic fibroblast growth factor (bFGF or FGF-2), an epithelial growth factor (EGF), a hepatocyte growth factor (HGF), an osteogenic factor (BMP) ), The soluble receptor is any one selected from the group consisting of a G-protein related receptor, a receptor tyrosine kinase, or an ionotropic receptor, or a combination thereof, Cain is an interleukin-2 (IL-2), interferon (INF) Kind-10, interleukin-1, interleukin-12, or chemokine, or a combination thereof. (I) A recombinant framework vector is prepared by inserting a polynucleotide encoding a human antibody Fc into a skeletal vector in a form capable of being expressed by a promoter of oriP originating from Epstein-Barr virus and a promoter of said skeletal vector step; Ii) operatively linking a polynucleotide encoding an exogenous protein linked to the candidate leader sequence peptide to a polynucleotide encoding a human antibody Fc of said recombinant framework vector to produce a recombinant expression vector; Iii) a control expression vector containing a polynucleotide encoding a fusion protein in which an indicator leader sequence is linked to the recombinant expression vector and the foreign protein in place of the candidate leader sequence peptide, respectively, is transfected into a host cell to prepare an experimental group and a control host cell ; Iv) culturing the test group and the control host cells to recover respective culture supernatant; V) quantifying the expression amount of an exogenous protein using an antibody specific for Fc; And Vi) screening the leader sequence for high-throughput production of the exogenous protein, comprising the step of selecting candidate leader sequence peptides that increase the production of foreign proteins in the experimental group as compared to the control group. 19. The screening method according to claim 18, wherein the foreign protein is soluble receptor CD1D. 19. The screening method according to claim 18, wherein the indicator leader sequence is a leader sequence of an exogenous protein itself, an alfalfa mosaic virus (AMV) RNA4 leader sequence, an invertase secretion signal of yeast or an acid phosphatase leader sequence of yeast. delete delete delete delete delete delete delete delete delete delete

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