CN1299871A - Human originating promoter for human body cell to express exogenous gene efficiently - Google Patents

Abstract

The present invention relates to a humanized promotor for human body cell to high-effectively express exogenous gene. In the concrete the invented promotor is from human thermal shock protein gene hsp 90 beta.

Description

The humanized's promotor that is used for the human body cell efficiently expressing exogenous gene

The present invention relates to a kind of humanized's promotor that is used for the human body cell efficiently expressing exogenous gene.Particularly, promotor of the present invention is from human heat shock protein gene hsp90 β, and described in this article promotor is called B6.1.

Since the second half in 20th century, very big variation has taken place in Human diseases spectrum.Along with the progress of molecular biology research and the expansion and the maturation of genetic engineering technique, constantly disclose the essence of disease, finds that the disease of new monogenic disease and multiple serious harm human health such as tumour, arteriosclerosis, hypertension, asthma etc. all have hereditary property change or the transgenation that polygene participates in.Therefore, use gene engineering product or directly carry out the necessary means that gene therapy has become the treatment human diseases.At present, but the polygonal virus expression systems of the intestinal bacteria of effective expression gene engineering product, yeast and insect often can not be finished the post-treatment of translating of various human proteinoid, therefore, is difficult to obtain the high biological activity product.And in mammalian cell owing to lack the corresponding strong promoter that efficiently expresses, foreign gene expression amount in vivo usually seldom, effectively biological activity is also very low.Be used for promotors such as the external cytomegalovirus that efficiently expresses of human cell, adenovirus, SV40 at present, and be usually used in foreign gene imported carriers such as retrovirus in the people's gene group, adenovirus etc. and all have possibility and cause that unexpected inherited character changes, produces than shortcomings such as dangerous more new virus of wild-type virus or potential carcinogenic risks.Therefore, make up a kind of non-viral, safety, Human genome strong promoter or carrier efficiently, will lay the foundation for degree of safety and the use range of widening gene therapy.

The known heat shock protein(HSP) of research in the past is the stress protein of a class high conservative, be present in nearly all organism, can be divided into following several big class: HSP104 according to its molecular weight size and homology, HSP90, HSP70, HSP60/GroEL, small molecules HSP (comprises HSP23,26,27 etc.) and ubiquitin etc.Heat shock protein(HSP) can different stress be as situations such as heat, heavy metal ion, virus infection, anoxics under abduction delivering, some HSP such as HSP90 also can express under normal physiological metabolism condition.Heat shock protein(HSP) plays an important role to the physiological processs such as protection of pair cell under normal physiological metabolism and stressed condition.In cell, HSP participates in Protein Folding, the assembling of subunit, and processes such as transportation and protein degradation are regulated target protein in the born of the same parents activity and function, the while does not participate in the composition of target protein again.Therefore, heat shock protein(HSP) be otherwise known as " molecular chaperones " (Molecular Chaperone).

Discover that the HSP90 gene is the member of containing intron few in number in the HSP family, it has α and two copies of β (Hickey, Mol.Cell Biol. (1989) 9:2615-2626 such as E; Rebbe, NF etc., J.Biol.Chem. (1989) 264,15006-15011) this gene not only be heated abduction delivering and also be high constructive expression (Shen YF, J.Cell.Physiol. (1996) Suppl.4,36-41).The inventor finds when research people quasi-lymphocyte hsp90 gene transcription regulatory mechanism, contain hsp90 β gene 5 ' upstream, (' total length ' 1039/+1531bp) (2.57kb) regulation and control fragment has composition and thermal induction expression activity (the Shen YF etc. that make the reporter gene high level expression for first exon and first intron, FEBS Lett. (1997) 443:92-98), its constructive expression's activity is higher than commercial in the world some viral promotors, as the regulation and control fragment of hsp90 β in lymphocyte than cytomegalovirus (cytomegalovirus, CMV) promoter activity is high 1 times, higher 5.8 times than simian sarcoma virus SV40 early promoter, only be lower than fowl sarcoma virus (Rous sarcoma virus, RSV) promoter activity slightly.Recent findings is in the bacterium tet-on carrier system of the efficient controlled expression of commercial external eukaryotic gene, and under its best inductive condition, gene expression efficiency also only is 3.9 times of the expression of gene level that is subjected to hsp90 β regulation and control fragment mediation.Therefore, people hsp90 β gene regulating fragment might be as the novel non-viral promotor that efficiently expresses goal gene in the body.And after external hyperthermic treatment, the activity of hsp90 β gene promoter can raise 2-2.5 doubly again.Show it might make goal gene occur temperature control down the ultra-high efficiency expression and be applied to clinical.In addition, hyperthermic treatment can make cell endogenous HSP90 and other molecular chaperones such as HSP70, and HSP60 etc. are abduction delivering, thereby helps the correct folding and transhipment of exogenous gene expression product, has higher biological activity to guarantee product.

In view of also existing negative regulatory element to influence the expression level of goal gene in the above-mentioned 2.57kb gene regulating fragment, therefore, the inventor is from human hsp90 β gene regulating fragment, carry out high constructive expression's optimization reorganization, finally obtain one section and had more highly active constructive expression's promoter fragment, finished the present invention thus.

Therefore, the object of the present invention is to provide a kind of promotor that is used for the human body cell efficiently expressing exogenous gene, this promotor is the people source, has overcome the shortcoming of viral promotor in the prior art.

According to the present invention, a kind of non-viral promotor that is used for the human body cell efficiently expressing exogenous gene is provided, called after B6.1, its length is 1738bp, its nucleotide sequence is shown in SEQID NO:1, in SEQ ID NO:1 ,+1 is transcription initiation site, and underscore represents CAAT box (87/-84) and TATA box (position 27/-24) respectively.

The length of promotor B6.1 of the present invention is 1738 bp, and its nucleotide sequence is seen SEQ IDNO:1, its kept hsp90 β gene from 5 ' upstream one 1039bp to the first intron+531bp fragment and first introne 3 ' end+1373/+1531bp.This promotor except that contain CAAT box (87/-84bp), the SP1 binding site (51bp), core promoter TATA box (27bp), transcription initiation site (+1bp), contain also that the inventor at first finds to the high composition of hsp90 β gene and thermal induction transcribe first introne 3 ' end of playing a crucial role be rich in the A/T district (+1373/+1531bp), and several transcription initiation sub-elements wherein.

As described in detail in the following embodiments; for obtaining promotor B6.1 of the present invention; the inventor has made up chloramphenicol acetyltransferase (the Chloramphenicol Acetyltransferase that dozens of contains the different regulating and controlling sequences of hsp90 β gene; CAT) or luciferase (Luciferase; Luc) reporter gene plasmid; and transfection Jurkat eukaryotic cell lines respectively repeatedly; be subjected to the cell of the reporter gene plasmid of different regulating and controlling sequences mediations by having detected transfection; corresponding enzymic activity in the cell pyrolysis liquid before and after heat-shocked has further determined to regulate and control composition in the hsp90 β gene and thermal induction is just transcribed; negative regulatory element and sequence thereof.Then according to the gained result, the controlling element of hsp90 β gene has been carried out repeatedly transitional reorganization, successively made up 16 of reporter gene plasmids that are subjected to the mediation of Different Optimization reorganization regulation and control fragment.Relative activity through the examining report gene filters out a recombinant plasmid pB6.1 that the constructive expression is the highest.The constructive expression of pB6.1 is contained 1.82 times of the segmental plasmid of above-mentioned hsp90 β gene 2.57kb gene regulating, and is not only strong than CMV and SV40 promotor, and surpasses the activity (seeing embodiment 2) of RSV promotor.

Therefore, compare, because promotor of the present invention is the humanized, so have safe advantage with the promotor of prior art.In addition, promotor energy mediate foreign gene of the present invention high level expression in the human cell, its intensity surpasses the viral promotors that was detected.

Promotor of the present invention can be used in the gene therapy, promptly by the exvivo gene transfer method, patient's blood is taken out, external will contain strong promoter of the present invention and be subjected to the plasmid transfection cell of goal gene of its regulation and control after, carry in the ex vivo again.Perhaps also can be by reaching the purpose of whole body or topical therapeutic in approach such as intramuscular injection or the respiratory tract suction input body.

Hereinafter with reference to drawings and Examples the present invention is described in more detail.Wherein

Fig. 1 is the synoptic diagram of hsp90 β gene ' total length ' regulation and control fragment.

Fig. 2 is the structure schema of plasmid pHSP90 β 1.

Fig. 3 is the structure schema of plasmid pHSP90 β 2.

Fig. 4 is the structure schema of plasmid pHSP90 β 3.

Fig. 5 is the structure schema of plasmid pHSP90 β 3.1.

Fig. 6 is the structure schema of plasmid pHSP90 β Bg.

Fig. 7 is the structure schema of plasmid pHSP90 β 6.1.

Fig. 8 illustrates B6.1 promotor and other 15 kinds based on the recombinant promoter specific activity of B3.1.

Fig. 9 is that the specific activity of B6.1 promotor and hsp90 β gene 2.57kb regulation and control fragment and four kinds of viral promotors is than synoptic diagram.

The structure 1. that embodiment 1 contains the reporter gene plasmid pHSP90 β 6.1 of promotor B6.1 contain hsp90 β gene 5 ' upstream fragment (the structure 1.1 hsp90 β gene 5 ' upstream fragments of plasmid pHSP90 β 1 1102/+67bp) (amplification 1102/+67bp):

The sequence (Fig. 1) of the people hsp90 β gene of delivering according to document, having designed and synthesized to increase contains hsp90 β gene core promotor, upstream starting element, cAMP response element, an atypia heat-shocked element (HSE,-648/-734) and segmental a pair of primer P1 of part first exon and P2, described primer sequence is as follows:

P1:5 ' GC ^-1102GAGCTCCGGCTGCCCTGCAC ^-10833 ' (underscore is Sac I site)

P2:5 ' GCGAATTC ^{+ 4}6GCAACGTAGGCTTGCTTTCCGA ^{+ 67}3 ' (underscore is EcoR I site) used this to primer, goes out the dna fragmentation of the 1.1kb consistent with expection from the DNA of human peripheral leucocytes λ GEM-11 genomic library (Promega company) through pcr amplification.1.2 hsp90 β gene 5 ' upstream fragment (clone 1102/+67bp):

Reclaim the 1.1kb pcr amplified fragment that is obtained, handling with the T4 archaeal dna polymerase successively makes fragment two hold endization level with both hands, EcoR I enzyme is cut, and the T4 polynueleotide kinase is handled and made 5 ' end phosphorylation, and pGEM-4Z carrier (Promega company) last and through EcoR I and Hind II double digestion is connected.Connect mixture transformed into escherichia coli XL-1blue bacterial strain, picking positive colony, preparation plasmid.Identify that through restriction enzyme enzyme spectrum analysis, Southem blot hybridization and order-checking confirmer hsp90 β gene-1 102/+67bp fragment has been directed the clone, this recombinant plasmid is named as pHSP90 β 1.The whole building process of this plasmid as shown in Figure 2.2. contain hsp90 β gene first exon and the first intron fragment (structure of plasmid pHSP90 β 2 2/+1531bp)

According to the sequence of the people hsp90 β gene of delivering, second couple of primer P3 and P4 have been synthesized in design, and its sequence is as follows:

P3:5 ' ^-2GTAGCTCTCTCGAGTCACT ^{+ 17}3 ' (underscore is Xho I site)

P4:5 ' ^{+ 153}0AAAATAAAAATCTCATTAAT ^{+ 1511}3 ' (underscore is Vsp I site) utilizes this to primer, with the human peripheral lymphocyte genomic dna is template, go out the 1530bp fragment through pcr amplification, from sepharose, reclaim this amplified fragments, after transition vector pCR II (Invitrogen company) is connected, transformed into escherichia coli XL-1blue bacterial strain, picking positive colony, preparation plasmid.Identify that through restriction enzyme enzyme spectrum analysis and order-checking confirmation hsp90 β gene first exon and the first intron fragment (2/+1531bp) are cloned in the transition vector.The gained plasmid is partially digested with Pst I complete degestion and Xho I, and recovery is inserted fragment and is cloned in the pBS-KS plasmid (Strategene company), is built into plasmid pHSP90 β 2 (Fig. 3).3. the structure of plasmid pHSP90 β 3

Earlier with EcoR I and Pst I digested plasmid pHSP90 β 1, to contain hsp90 β gene 5 ' upstream fragment and (1039/+67bp) insert pBS-KS, then, with this transition plasmid of Xho I complete degestion, recovery contains the big fragment of hsp90 β gene-1 039/+7bp, after calf intestinal alkaline phosphatase is handled, recombinate with fragment that reclaim, that contain hsp90 β gene+7/+1531bp behind the partially digested pHSP90 β 2 of Xho I, be built into plasmid pHSP90 β 3 (Fig. 4).This plasmid contains hsp90 β gene-1 039/+1531bp fragment.4. the structure of chloramphenicol acetyltransferase (CAT) reporter gene plasmid pHSP90 β 3.1 and pHSP90 β Bg

Use Pst I complete degestion and the partially digested plasmid pHSP90 of Xho I β 3 successively, recovery contains the fragment of hsp90 β gene-1 039/+1531bp, insert between the corresponding multiple clone site of CAT reporter gene plasmid pBLCAT3 (Jia Hong professor of Beijing Medical University gives), be built into plasmid pHSP90 β 3.1 (Fig. 5) .Then, reclaim big fragment, from connecting, be built into plasmid pHSP90 β Bg through the T4 dna ligase with Bgl II digested plasmid pHSP90 β 3.1.Compare with pHSP90 β 3.1, deleted in the CAT upstream region of gene regulating and controlling sequence of pHSP90 β Bg in hsp90 β gene first intron+the 110/+1372bp fragment, therefore, with in pHSP90 β 3.1 plasmids+the 1373/+1531bp fragment inserts fragments sequence and number changes into+109/+268bp (Fig. 6) .5. the structure of plasmid pHSP90 β 6.1

At first use hsp90 β gene+531bp site among the Mlu I digested plasmid pHSP90 β 3.1, after Klenow enzyme benefit is flat, cut-the 1039bp site with Pst I enzyme again, reclaim small segment (being hsp90 β gene-1 039/+531bp fragment).In addition with Bgl II digested plasmid pHSP90 β Bg, through the Klenow enzyme mend flat after, cut-the 1039bp site with Pst I enzyme again, reclaim big fragment and (wherein contain+the 109/+268bp fragment, be equivalent in the hsp90 β gene+1373/+1531bp).Then, two fragments that reclaim are connected, be built into plasmid pHSP90 β 6.1.Compare with plasmid pHSP90 β 3.1, pHSP90 β 6.1 has deleted 840bp in hsp90 β gene first intron, and (+532/+1372bp) fragment, inserting fragment length in this plasmid is 1738bp (Fig. 7).Hsp90 β gene regulating fragment among the pHSP90 β 6.1 is named as promotor B6.1, and its sequence is seen shown in the SEQ ID NO:1.The mensuration of embodiment 2 B6.1 promoter activities and with the mensuration 1. plasmid transfection [Ausubels of other viral promotors specific activity than A. method B6.1 promoter activity; F.M.; Brent; R.; Kinston; RE.; et al.; Current protocol in molecular biology; New York; Greene PublishingAssociated and Wiley-Interscience; 1987.] extracting chloromycetin acetyltransferase (CAT) reporter gene plasmid pB6.1 and pHSP90 β 3.1 or other each 9 μ g of optimization recombinant plasmid based on hsp90 β gene 2.57kb regulation and control fragment (B3.1); add STBS damping fluid (25mM Tris-HCl pH7.3,137mM NaCl, the 6mM Na that contains 500 μ g DEAE-Dextran (Pharmacia company) respectively ₂HPO ₄, 0.7mM CaCl ₂, 0.5mMMgCl ₂) Jurkat cell (2 * 10 ⁷) in the suspension, and add 1 μ g beta-galactosidase enzymes (β-Galactosidase, the reporter gene plasmid pSV-β-gal (Promega company) of β-Ga1) is as the transfection efficiency internal reference, 37 ℃ cultivate 90 minutes after, drip DMSO to final concentration 10%, wash cell 3 times with STBS after 2 minutes, again cell grouping changed over to 12 well culture plates, 37 ℃ cultivate 66 hours after collecting cell.

2. the active mensuration [Sambrook of chloramphenicol acetyltransferase; J.; Fritsch; E.F.; Maniatis; T.; Molecular Cloning:A Laboratory Manual 2nd ed:NewYork, Cold Spring Harbor Laboratory, 1989.] transfectional cell is after PBS washes 2 times; be suspended in the cell pyrolysis liquid (250mM Tris-HCl pH7.8); multigelation is 3-4 time in liquid nitrogen and 37 ℃ of water-baths, then, and at 4 ℃; 12000 rev/mins centrifugal 20 minutes, collect supernatant liquor.After in 30 μ l supernatant liquors, adding 20 μ l 100mM Tris-HCl pH7.8 solution,, add 200 μ l reaction solutions (100mMTris-HCl pH7.8,125mM paraxin, 0.1 μ Ci more successively 65 ℃ of heating 15 minutes ³The acetyl-CoA of H mark (Amersham company)) and the 5ml scintillation solution, after 12-14 hour, measure the cpm value of each sample 37 ℃ of reactions with Liquid Lumex instrument.Then, proofread and correct with the galactosidase activity in the respective sample again.

3. the mensuration of betagalactosidase activity adds 40 μ lLumiGal reaction solutions (Clontech company) in 6 μ l lysis supernatant liquors, at room temperature reaction after 1 hour, measures fluorescence intensity in each supernatant liquor with Monolight 2010 luminoscopes.The mensuration of active mensuration 1. plasmid transfections of several viral promotors and uciferase activity respectively will be with fowl sarcoma virus (RSV) long terminal repeat (LTR), cytomegalovirus (CMV) promotor and hsp90 β gene " total length " fragment (1039/+1531bp) for three kinds of luciferases (Luc) reporter gene plasmid of promotor (making up) by this group and be subjected to simian sarcoma virus SV40 early promoter and the Luc plasmid pGL2 (Promega company) of enhanser regulation and control with DEAE-Dextran method (seeing aforesaid method) or Lipofectamine method (Gibco-BRL company) transfection Jurkat cell, 37 ℃ cultivate 48 hours after, collecting cell, in each group cell, add 100 μ l lysates (Promega company), after room temperature is placed 15-20 minute, at 4 ℃, 12000 rev/mins, centrifugal 20 minutes, collect supernatant.Get 20 μ l supernatant liquors, measure fluorescence intensity in each supernatant liquor with Luciferase Reaction System (Promega company) and Monolight 2010 luminoscopes.Proofread and correct with the protein concentration of respective sample again.2. the mensuration of protein concentration is substantially according to the Coomassie Brillent BlueG-250 development process [Bradford of Bradford, M.M., Concentration of Protein:CoomassieBlue Solution.Anal Biochem, 1976,72:248.] measure the total protein concentration of cell pyrolysis liquid.The promoter activity of B6.1 as a result one:

Transfection has the CAT reporter gene plasmid pHSP90 β 6.1 of B6.1 and contains in the human lymphocyte of pHSP90 β 3.1 of " total length " 2.57kb CAT activity relatively more respectively, activity with the promotor of pHSP90 β 3.1 is 100 o'clock, in pHSP90 β 6.1 plasmids, CAT activity under B6.1 drives is 183.8+42.3, promptly being higher than " total length " fragment more than 1.8 times, is in 15 kinds of recombinant plasmids that make up the same period and detect active the highest (Fig. 8).Two, the active comparison of B3.1 and viral promotors

The uciferase activity that will contain the luciferase reporter gene plasmid of hsp90 β gene " total length " fragment (B3.1) compares with the uciferase activity that is subjected to 3 kinds of commercial viral promotors mediations respectively.With the segmental promoter activity of hsp90 β gene " total length " is 100, finds that the CMV promoter activity is 48.3% of hsp90 β, and the SV40 early promoter is its l7.1%, has only the RSV promoter activity to be higher than segmental 1.60 times of hsp90 β gene " total length ".

Comprehensive relatively The above results finds that the B6.1 promoter activity is higher than all viral promotors that detected (Fig. 9).

The information of sequence table SEQ IDNO:1: sequence signature:

(A) length: 1738bp

(B) type: nucleic acid

(C) chain: two strands

( D ) ：：SEQ ID NO:1-1044 CTGCAGGAGC GAAGTGGGCG GAAAAAAAGC GAACCAGCTT GAGAAAGGGC-994 TTGACGTGCC TGCGTAGGGA GGGCGCATGT CCCCGTGCTC CGTGTACGTG-944 GCGGCCGCAG GGGCTAGAGG GGGGTCCCCC CCGCAGGTAC TCCACTCTCA-894 GTCTGCAAAA GTGTACGCCC GCAGAGCCGC CCCAGGTGCC TGGGTGTTGT-844 GTGATTGACG CGGGGAAGGA GGGGTCAGCC GATCCCTCCC CAACCCTCCA-794 TCCCATCCCT GAGGATTGGG CTGGTACCCG CGTCTCTCGG ACAGGTCAGA-744 GCGGGTCGCC GGGTGGGGTC GCTGCAAAAA CCCTGCCCCG GCCGCAGCCG-694 AGAGGCGGAC GTCGCGGGGA GGGGGCGGGA CCGCCGAGAC AGGCCTGGAA-644 ACTGCTGGAA ATGCCGCAGT GCCGCCGCCG CCCCTTCCGC CGCATGTCGG-594 CAAAGAGTCC CCGCCAGCCC CGGCCGGCGC CCTCCCCCTA CGCTGAGCTG-544 CCCCTCAGCG CGAACCCTCC GCCCTTTCCT CTACTCTTGC GAGAGTCGGG-494 ATCTGGGGCT ACCCAAGGTT GGGTCCCGAA TGCCAGTCCC TCTGTCGGGA-444 CGCGAGATGT GTAGGGCAGA TGCTAGGAAG AAGATTGGGT CTGGGAGCGG-394 TGGTCCGCGT GGTTAGCTGC CTCCGCTCTT TTTCGGTGTC CCCCCCAGTC-344 CCGCCCTTGG GTGTGGGGAC GCCTGCCCCA CAAGTGTTTA GGGAGGTCAG-294 TGGGTTCCTC GCCCGTAGAG ACTCCGTTTA TGCCAAATGA GCACTCCTCA-244 TCCCCGCTCT TGATGGAGTC ATGTCCTAGA CGTGAAACTA TGGGGCTGTG-194 ATCACAAGCA AATGTGTGGG CGGATCCGTT GCTTGGGTTC TTCCCCGCCC-144 CCTCCTTTTT TCGGACCATG ACGTCAAGGT GGGCTGGTGG CGGCAGGTGC-94 GGGGTTGACA ATCATACTCC TTTAAGGCGG AGGGATCTAC AGGAGGGCGG +1-44 CTGTACTGTG CTTCGCCTTA TATAGGGCGA CTTGGGGCAC GCAGTAGCTC+7 TCTCGAGTCA CTCCGGCGCA GTGTTGGGAC TGTCTGGGTA TCGGAAAGCA+57 AGCCTACGTT GCTCACTATT ACGTATAATC CTTTTCTTTT CAAGGTAAGG+107 CTGAGATCTC CGCTAGCCTT CTTTCCCTTT AGTGCTGTAT TCGTGTTGTT+157 TTTGTTTTTT TCTGTCCTTT AGGGAGCCTT AGTCTAGATG TCGGGGTGGC+207 TTGTGGATAA CGCTCTGGAT TTTTATAGGG TGAGGGTAGT GGTGGGTGAG+257 GTTTTTTGAG TCCTCCTCGG TTTTCTCTAG TGTGTTTGGG GGGTGGGGCT+307 TTCTCTCGGC GCCTGCTGGC CGTAGCGAGG TGGGCTGTGG GGTTGGGGCA+357 GTGGGCGGCT GGCAGCTGCA CGTGGTGGCC GCGCGGCCCG GGACGCTGCC+407 ATTTTTGCCC CTCCACTTCC GGACGCGGCT ACGGGGCGTC GGAGGGGGAC+457 CGCAGGGTGG CGGGGGTGCC CGCTGGGGTG ACTCAGCACG GCCTTGTGGG+507 ACTGGCTTTG TCACCTCTCT TATCGGACGC GGATCTTTAA GAGAATGCAT+557 TTTTAGTCTT GGGAAGGGAT AGTACTCCGG TTAAACCAGT CTGAACTCAC+607 TGTCTAAGGT CCTAACAAAT GATATGACCT TTAGGATTTT TAAACATGGG+657 GCCTTAGTGT TCTTTTGTAA TTAATGAGAT TTTTATTTT

Claims (1)

1, a kind of promotor, it is from HUMAN HEAT SHOCK PROTEINS gene hsp90 β, and its nucleotide sequence is shown in SEQ ID NO:1.

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