Method of producing fragment of nucleic acid encoding human proinsulin

专利摘要:
A DNA having a base sequence coding for human proinsulin and a DNA having a base sequence coding for human pre-proinsulin have been cloned, and novel recombinant DNA transfer vectors containing said cloned DNAs have been constructed. Novel microorganisms transformed by said recombinant transfer vectors have been obtained. Certain of said transformed microorganisms have demonstrated capability to express the cloned DNA's, synthesizing a protein comprising human proinsulin and a protein comprising human pre-proinsulin.
公开号:SU1491345A3
申请号:SU802994154
申请日:1980-09-11
公开日:1989-06-30
发明作者:Белл Грем；Луис Пиктет Рэймонд；Майкл Гудман Говард；Дж.Раттер Вильям
申请人:Дзе Риджантс Оф Дзе Юниверсити Оф Калифорния (Фирма)；
IPC主号:

专利说明:

The invention relates to the field of genetic engineering and concerns the preparation of a nucleic acid fragment encoding human proinsulin using recombinant DNA technology.
The DNA fragment encoding proinsulin is obtained by cloning a mammalian gene. After cloning in the plasmid pBR322, it is isolated using the Hhal and Alul endonucleases and the chemically synthesized 3 -TTTGTGAAGGAAGACACCTGTGGGG CTCAGAGCTGGAAG-5 amino acid nucleotide sequence encoding 14 amino acids of proinsulin is modified to attach.
Example 1. RNA is obtained from human insulinoma after centrifugation in 5.7 M CsCl containing 100 mM EDTA, and used without further fractionation as template dp for cDNA synthesis using reverse transcriptase and SI nuclease. Approximately 40 ng of the double-stranded cDNA is obtained from 130 μg of eefracdionated RNA.
The unfractionated cDNA is treated with a terminal transferase in the presence of dCTf to produce oligo-C tails at the 3 ends of the cDNA molecules. Plasmid pBR322 is digested with endonuclease restriction Pst I and digestion P
 4 ate

sn
heat the transferase in the presence of dGTP to obtain oligo-dG-xBOCTOB at the 3 ends of the linear plasmid DNA.
Sew the obtained fragments and get the circular plasmid.
1 1tam E.coli XI776 is transformed using plasmid DNA-containing inserts. 525 tetracycline-resistant transformants are obtained, they are replicated by seeding and classified into insulin sequences using hybridization of colonies (in situ) i The previously cloned rats of preproinsulin rats are used as samples for hybridization, after the label of rats cDNA using Nick-translation using DNA polymerases 1c. Since the amino acid sequences of insulin in rats and humans are quite similar (insulin, 92% gomlog; proinsulin, 83% gomlog), it is suggested that the cloned rat cDNA will test cc hybridization with human insulin sequences under non-rigid conditions. Autoradiography reveals that two out of 525 colonies are hybridized with a sample of cloned prepro insulin-1 rats. Both colonies are sensitive to ampicillin. Plasmids isolated from colonies are approximately 250 base pairs (Lp) and 500 Lp longer than pBR 322 itself. The longer plasmid is designated pcH1-1.
The nucleotide sequence of the inserted cDNA fragment pcH1-1 is determined by the method of Makhash and Gilbert
Primer 2o cDNA insert pcH1-1 contains the complete coding sequence for human front-ninsulin on
DNA synthetic proisisulin
treated with Alu-1-endonuclease to obtain two fragments of size A3 Lp and 56 Lp. Similarly, the pCH1-1 cDNA insert, preferably obtained by treatment with Hh a I, is cleaved by partial hydrolysis catalyzed by Alu-1-endonuclease to form fragments of 75, 90, 110, 135, 165, 200, 245, 275, 375 and 455 Lp, respectively, are fractionated by gel electrophoresis to obtain a fragment of 375 Lp, which is obtained by breaking in one location in the codon for amino acid N 14 o
0
five
0
five
0
five
0
five
0
five
The fragments of the synthetic gene and the frant ment of 375 Lp are joined using a blunt ligature. The exact combination of a 43 Lp synthetic fragment with a 375 Lp cDNA fragment is maximized, provided that 375 Lp cDNA is present in a molar excess. Opportunities for inaccurate connections are also minimized by the fact that the synthetic fragments have single stranded protruding ends that are not complementary to the ends of the 375 lp cDNA fragment.
The combined fragment containing the synthetic sequence for methionine (1-13 amino acids) and the naturally occurring coding sequence for amino acids 14-86 of proinsulin constitute the coding sequence for proinsulin.
The proinsulin-coding sequence is designed by selectively breaking the internal position in the proinsulin-coding region, followed by stitching a chemically synthesized sequence that encodes that part of the proinsulin-coding region that was removed by previous cleavage. Plasmid pcN1-1 is used as a source of proinsulin-coding region, which is selectively excluded by treatment with Hhal-endonuclease
After isolation, any fragment is treated with alkaline phosphatase to remove the 5-terminal phosphate groups, then digested with Alu I endonuclease, which has a unique cleavage point in the 13-14 amino acid region in the pro-sulin-coding sequence. The location of the restriction is preferably located near one of the ends of the proinsulin-coding sequence. The Hhal fragment - pcH1-1 is partially broken by the Alul-endonuclease to produce two fragments of approximately 76 Lp and 375 Lp, respectively. vetstvenno. Alul fragments are fractionated by gel electrophoresis and a fragment of 375 Lp is isolated.
The nucleotide sequence encoding the first 13 amino acids of proinsulin with 5-terminal C (plus-strand), to terminate the codon for alanine at position 14, synthesize phosphotri5I
by the ethereal method of the Plus-thread synthetic DIC HN: em sequence: 3 -TTTGTGAACCAACACCTGTGCGGCTCACACCTG GTGGAAG-5. corresponding to the natural sequence,
The resulting sequence is
sewn with a blunt end with approximately 375 Lp fragment of the Hhal fragment of pcH1-1. Since the latter has a 5 -phosphate only at the attached end, the two fragments are joined in the correct order. The synthetic fragment is properly combined with a large fragment in approximately 50% of the reaction.


-24
-...20
1, Method for producing a fragment of n 15 kleinic acid encoding a human proline with the following nucleotide sequence: -10
met ala ei trp met org teu leu pro leu leu ofa leu ola let / CCTTCTCCCATD CCC CTC TCC PBX CCC CTC CTC CCC CTC CTC CCC CTC CTC CCC CTC
Example 3. The cloned inserts encoding preproinsulin (example 1) or proinsulin (example 2) are inserted into an expression transfer vector. When an insertion occurs in the correct orientation, relative to the start of translation at the insertion location, it is in phase relative to the read frame.

权利要求:
Claims (1)
[1]
Invention Formula
1, Method for producing a fragment of a nucleic acid encoding a human proinsulin with the following nucleotide sequence: -10
.trp gttf
TCC CCA
ten
pro ohr pro ofa ofo afo pfie vol oia gfn h / 5 teu cy ser his feu vat
CCT CAC CCA CCC CCA CCC TTT CUC AAC CAA CAC CTC / CC CCC tCA CAC CT & CT & SDA
Qlo teu gr
CCT CTC111 N r
Alu f 20JO
ti / r feu vof ci / 5 gig glu orggfy p / ie phe tj / r thr pro lys thrarg org gig alo
TAC СТА CTt TCC CCC CAA CCACCC TTC TTC AC ACAC CCC ACCCC CCC CAC CCA
glu asp CAC CAC
0 so
leu gin wl gig val fi gig g / y gfc / pro gfi / olo gig str feu gfn pro
C & C CAC & TC CAC CG CTC CG CTC CG CTC CG CTC CG CTC CG CTC CG CTC
eu ofo
TT & GCC
Aluf
60 70
leu glu gfv ser leu gin tyj arg gtg lie ko glu gin tgi cys thr ser lie cyi
CTC GAD CCC TCCCTD CAG AAG CCT CCC ATT GTC GAA GARDEN TCC TGT ACC AGO PBX TCC
ser leu.
Tcc hundred
60th
tyr gfn leu g / uasn tyr cyj 05P
TAC CAC CTG CAGAAC TAC TGC AAC TAG ACGCACCCCCCACCCACCCCCA CCCCCCCCCTCCTCCA
Aih
w / ffj
CCCACA & ACATDGAATAAACCCCTTCAACCACC
CM I
that is, cells of Langerhans islet center}) are uglirovanny in a solution containing 5.7 M CsCl and 100 EDTA, RNA prepro-insulin is separated, cDNA is synthesized, the resulting cDNA is inserted into the PstI plasmid pBR 322 site using dA- oligo-dT-method, the strains of Escherichia coli bacteria are transformed with recombinant plasmid DNA and the clones hybridizing from the cloned cDNA of preproinsulin in rats containing the radioactive label are selected, the plasmid pcN1-1 is inserted with the pre-insulin cDNA insertion, treated with p1H1. Hha I endonuclease followed by frequent chnym gi; Roliz Alul zndonukleazoy.
5c
a fragment of 375 Lp in length is isolated and ligated with a DNA ligase T with a fragment of 43 Lp in a chemically synthesized nucleotide sequence 3 -TTTGTGAACACACACC TGTGCGGCTCACACCT GGTGG AAG-5 encoding 1-14 amino acids of proinsulin, ending with Alu lo
Po1, distinguished by the plasmid pcN1-1
2, the method according to the n and with that, is treated with the Hhal nucleonuclease, a fragment of 375 lp in length is isolated, which are bluntly ligated with the nucleotide sequence 3 -TTTTGT GAACCAACACCTGTGCGGCACACACGGTGGAAG-5.

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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US7519279A| true| 1979-09-12|1979-09-12|

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