WO2017152808A1 - 利用优化的基因密码子扩展系统通读提前终止密码子疾病中的截短蛋白 - Google Patents

利用优化的基因密码子扩展系统通读提前终止密码子疾病中的截短蛋白 Download PDF

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WO2017152808A1
WO2017152808A1 PCT/CN2017/075577 CN2017075577W WO2017152808A1 WO 2017152808 A1 WO2017152808 A1 WO 2017152808A1 CN 2017075577 W CN2017075577 W CN 2017075577W WO 2017152808 A1 WO2017152808 A1 WO 2017152808A1
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trna
amino acid
protein
natural amino
smad
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夏青
杨琦
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Peking University
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Definitions

  • the invention belongs to the field of biopharmaceutics, and particularly relates to a non-natural amino acid system which utilizes gene code expansion, and reads through a nonsense mutation site of a single gene genetic disease. And by modifying the tRNA (tRNAPyl) of M. oxysporum, a new high-through-reading UAA and UGA-encoded unnatural amino acid system was obtained, extending the range of use of tRNAPyl and pyrrolysyl-tRNA synthetase (PylRS) orthogonal pairs. It can be used to read through the stop codons UAG, UAA and UGA of three nonsense mutations.
  • tRNAPyl tRNA
  • PylRS pyrrolysyl-tRNA synthetase
  • nonsense mutations are one of the gene mutations.
  • Gene mutations are heritable variations in genomic DNA molecules, including frameshift mutations and base substitutions.
  • Frameshift mutations include insertions and deletions of bases, which are mainly missense mutations and nonsense mutations.
  • a nonsense mutation refers to the mutation of a certain base of the coding gene, resulting in the stop codons UAG, UAA and UGA, and the stop codon does not encode any amino acid.
  • the stop codon cannot be paired with the anti-codon of the transfer RNA (tRNA), but can be recognized by the terminator or release factor, terminating the synthesis of the peptide bond, terminating protein synthesis, and thus producing an incomplete and non-functional protein.
  • PTC Premature termination codons
  • Duchennemuscular dystrophy is a typical representative of PTC disease.
  • DMD is a serious muscle atrophy disease and the most common X-linked recessive hereditary disease. It is characterized by progressive and lethality.
  • DMD base The nonsense mutation is one of the main causes of DMD.
  • the nonsense mutation produces a premature stop codon UAG, UAA, UGA, resulting in a truncated polypeptide product that causes the patient to lack or lack functional dystrophin, resulting in muscle atrophy.
  • the incidence of Duchenne muscular dystrophy in live births is 1/6300 to 1/3500 [Dooley J, Gordon KE, Dodds L, MacSween J.
  • Duchenne muscular dystrophy a 30-year population-based Incidence study. Clin Pediatr (Phila), 2010, 49: 177-179.]. There is no effective cure for this disease, more than the onset of childhood, loss of walking ability in adolescence, death in early adulthood, and a heavy psychological and economic burden on patients, families and society.
  • Inhibitor tRNA read Its anti-codon loop is mutated and can be paired with a stop codon so that the stop codon can be read through. .
  • the main reason that this treatment is difficult to enter clinical applications is that the suppressor tRNA may recognize a normal stop codon resulting in the potential toxicity of the abnormal protein.
  • the non-native aminoacyl tRNA synthetase is an aminoacyl tRNA synthetase from a microorganism such as archaea or Escherichia coli, which has been modified by positive and negative screening artificial protein sequences and has no specific sequence to bind to endogenous tRNA of E. coli or eukaryote.
  • Aminoacyl tRNA synthetase References: Wang L, Schultz P G. Expanding the genetic code [J]. Angewandte chemie international edition, 2005, 44(1): 34-66.
  • orthogonal of tRNA and non-native aminoacyl tRNA synthetase as used herein means that this tRNA is not a substrate for any endogenous aminoacyl tRNA synthetase, and this aminoacyl tRNA synthetase cannot be aminoacylated. Any endogenous tRNA.
  • This orthogonal pair has a unique correspondence with each other. The meaning of orthogonality can also be referred to the literature: Wang L, Schultz P G. Expanding the genetic code [J]. Angewandte chemie international edition, 2005, 44(1): 34-66.
  • the invention relates to a tRNA, wherein the base CUA on the tRNA anticodon loop is mutated to UUA or UCA, and the mutated tRNA is further ligated by at least one non-native aminoacyl tRNA synthetase orthogonal thereto Identification.
  • the invention relates to a tRNA, wherein the anti-codon loop of the tRNA is not bound to at least one non-native aminoacyl tRNA synthetase orthogonal thereto.
  • the invention relates to a tRNA, wherein the tRNA is a tRNA derived from M. oxysporum (tRNAPyl), or a tRNA derived from E. coli, preferably the tRNA is tRNA Pyl UUA , the sequence of which is SEQ ID NO :1, or tRNA Pyl UCA , the sequence of which is SEQ ID NO: 2.
  • tRNAPyl M. oxysporum
  • E. coli a tRNA derived from E. coli
  • the invention relates to a non-natural amino acid system, wherein the system comprises a tRNA of any aspect of the invention and at least one non-native aminoacyl tRNA synthetase or nucleic acid sequence encoding the same, preferably, said
  • the non-natural amino acid system is selected from the group consisting of a lysine-like non-natural amino acid system, a leucine-based non-natural amino acid system, and a tyrosine-based non-natural amino acid system, optionally wherein the lysine-based non-natural amino acid system includes a source Since the tRNA (tRNAPyl) and pyrrolysyl-tRNA synthetase (PylRS) of Methanococcus, the leucine unnatural amino acid system includes tRNA (tRNALeu), leucyl-tRNA synthetase derived from Escherichia coli (LeuRS), the tyrosine-based non-natural amino acid
  • the invention relates to a non-natural amino acid system selected from the group consisting of:
  • Lys-diazirine NAEK
  • Lys-azido Shown Lys-diazirine (NAEK), Lys-azido shown, or
  • the non-natural amino acids having a diaziridine and an azide structure
  • the leucine unnatural amino acid being selected from the group consisting of: The Anap shown
  • the tyrosine non-natural amino acid is selected from the group consisting of: The pAcF shown.
  • the invention relates to a plasmid, vector, host cell or kit comprising a tRNA of any aspect of the invention or a non-natural amino acid system of any of the aspects of the invention.
  • the invention relates to a method of gene codon expansion, wherein a base CUA on a tRNA anticodon loop is point-mutated to UUA and UCA, and the mutated tRNA can also be synthesized by its corresponding unnatural amino acid tRNA Enzyme recognition.
  • the tRNA is a tRNA derived from M. oxysporum (tRNAPyl), or a tRNA derived from Escherichia coli, preferably the tRNA is tRNA Pyl UUA , the sequence of which is SEQ ID NO :1, or tRNA Pyl UCA , the sequence of which is SEQ ID NO: 2.
  • the invention relates to the use of a tRNA of any aspect of the invention or a non-natural amino acid system of any of the aspects of the invention, in the manufacture of a medicament for treating a genetic disease or cancer, wherein the genetic disease or cancer is due to a gene A nonsense mutation causes, preferably, the genetic disease or cancer is caused by a nonsense mutation in a Dystrophin protein, a tumor suppressor gene STK11 or an EPHB2 protein.
  • the genetic disease and cancer are selected from the group consisting of: Duchenne muscular dystrophy, cystic fibrosis, hemophilia A, hemophilia B, lipid storage, ataxia telangiectasia, Heraeus syndrome, family black idiots, stomach cancer, lung cancer.
  • the invention relates to a method of restoring normal expression and function of restoring a nonsense mutant protein, wherein a tRNA of any aspect of the invention or a non-natural amino acid system of any of the aspects of the invention is introduced into a non-sense mutant protein Cells or organisms.
  • a method of any of the aspects of the invention wherein the introduced tRNA or non-natural amino acid system recognizes a nonsense mutation of the protein of interest, and introduces a non-natural amino acid at a corresponding site of the nonsense mutation to allow translation of the protein of interest to avoid premature termination and Synthesize intact functional proteins.
  • the direction from R1 to R2 is the N-terminus to the C-terminal direction of the amino acid sequence, and the N-th position may be any amino acid on the pathogenic protein or the tumor suppressor gene protein, and correspondingly, R1 is the first to the N-th 1 amino acid residue, R2 is the amino acid residue from the N+1th to the C-terminus, R3 is or
  • the introduced unnatural amino acid is Lys-azido at the N-th position, and its linkage in the pathogenic protein or tumor suppressor protein is as follows:
  • R 1 to R 2 is the N-terminal to C-terminal direction of the amino acid sequence
  • the N-th position may be any one of the pathogenic protein or the tumor suppressor gene protein according to claim 1, correspondingly, R 1 is an amino acid residue at positions 1 to N-1, and R 2 is an amino acid residue at the N+1th to C-terminus,
  • the invention relates to a mammalian stable cell line which is HEK293-PYL, deposited on November 17, 2015, and deposited under the number CGMCC No: 11592.
  • a method for evaluating a gene codon extension technique characterized in that the read-through efficiency is evaluated by endogenously mentioning the amount of Smad protein expressed by the stop codon plasmid, preferably by the following steps:
  • mutant plasmids pcDNA3-Smad-39TAG, pcDNA3-Smad-122TAG and pcDNA3-Smad-133TAG are obtained, preferably the mutant sequences are as shown in SEQ ID NOs: 4-6;
  • the invention relates to a primer pair, wherein the primer pair sequence is PCMV-UAG-UAA-for:TGTAGATCGAATGGACTTTAAATCCGTTCCCCCGG and PCMV-UAG-UAA-rev:CCGGCTGAACGGATTTAAAGTCCATTCGATCTACA
  • PCMV-UAG-UGA-for CATGTAGATCGAATGGACTTCAAATCCGTTCAGCCGGGTT and PCMV-UAG-UGA-rev: AACCCGGCTGAACGGATTTGAAGTCCATTCGATCTACATG.
  • the invention relates to the use of an optimized gene codon extension technique to insert a non-natural amino acid at the premature stop codon of a nonsense mutant protein, and to read through the pathogenic protein and tumor cell suppression in a single genetic disease.
  • the invention relates to tRNA (tRNAPyl) of C. aureus, which is engineered from the anti-codon loop site-directed mutagenesis of the original tRNA Pyl CUA to tRNA Pyl UUA and tRNA Pyl UCA , the sequences corresponding to SEQ ID NO: 1 and SEQ ID NO, respectively. : 2, which is characterized by perfect pairing with the stop codons UAA and UGA, respectively.
  • tRNA Pyl UUA and tRNA Pyl UCA were constructed on PCMV-UUA and PCMV-UCA plasmid.
  • the invention relates to a pathogenic protein or a tumor suppressor gene protein, wherein the inserted non-natural amino acid is Lys-diazirine at the N-position, and the manner of attachment in the protein is as follows:
  • the direction from R 1 to R 2 is the N-terminus to the C-terminal direction of the amino acid sequence, and the N-th position may be any amino acid on the pathogenic protein or the tumor suppressor gene protein, and correspondingly, R 1 is the first to An amino acid residue at position N-1, and R 2 is an amino acid residue from the N+1th to the C-terminus,
  • the invention relates to a pathogenic protein or a tumor suppressor gene protein, wherein the introduced non-natural amino acid is Lys-azido located at the N-th position, and the linkage manner in the pathogenic protein or the tumor suppressor gene protein is as follows Show:
  • R 1 to R 2 is the N-terminal to C-terminal direction of the amino acid sequence
  • the N-th position may be any one of the pathogenic protein or the tumor suppressor gene protein according to claim 1, correspondingly, R 1 is an amino acid residue at positions 1 to N-1, and R 2 is an amino acid residue at the N+1th to C-terminus,
  • the invention relates to a gene codon extension technique, wherein the read efficiency is evaluated by endogenously raising the amount of the Smad protein expressed by the stop codon plasmid pcDNA3-Smad.
  • Mutant 39,122,133 codons were mutated into UAG amber stop codon, mutant plasmid pcDNA3-Smad-39TAG, pcDNA3-Smad-122TAG and pcDNA3-Smad-133TAG.
  • the sequences are shown in SEQ ID NOS: 4-6.
  • the mutant plasmid was transfected into the stable cell line HEK293-PYL.
  • the protein was extracted after 48 hours of non-natural amino acid culture, and the full-length Smad protein was detected by western blot.
  • the invention relates to a mammalian stable cell line stably expressing tRNA (tRNA Pyl CUA ) and pyrrolysyl-tRNA synthetase (PylRS), which is HEK293-PYL, deposited on November 17, 2015 The deposit number is CGMCC No: 11592. And a HEK293-PYL-TAA stable cell line stably expressing tRNA Pyl UUA / PylRS, and a HEK293-PYL-TGA stable cell line stably expressing tRNA Pyl UCA / PylRS.
  • tRNA Pyl CUA tRNA Pyl CUA
  • PylRS pyrrolysyl-tRNA synthetase
  • three tRNA Pyl /PylRS plasmids recognizing three stop codons (amber, ocher, opal) are constructed.
  • HEK293-PYL has been deposited in the China General Microbial Culture Collection Management Center, deposited on November 17, 2015, and the deposit number is CGMCC No: 11592. Its classification is human HEK293T cells) to restore DMD disease.
  • the expression of the protein Dystrophin, the endogenous premature stop codon and the expression of the tumor suppressor genes STK11 and EPHB2 were restored in the A549 and DU145 tumor cell lines.
  • PCMV-CUA tRNA Pyl CUA / PylRS
  • PCMV-CUA tRNA Pyl CUA / PylRS
  • the design point mutation primers, site-directed mutagenesis kit, the tRNA Pyl CUA anti base on the sub-ring password CUA point mutation is carried out by the above UUA UCA and primers to obtain PCMV-UUA (tRNA Pyl UUA / PylRS) and PCMV-UCA (tRNA Pyl UCA / PylRS) plasmid.
  • the read efficiency of three tRNA Pyl CUA/UUA/UCA /PylRS is detected using GFP green fluorescent protein containing a premature stop codon.
  • the 39th amino acid codon of GFP fluorescent gene was mutated to the three premature stop codons of UAG, UAA and UGA by point mutation technique to obtain pcDNA3.1-GFP-39TAG, pcDNA3.1-GFP-39TAA and pcDNA3. .1-GFP-39TGA three plasmids.
  • 293T cells were co-transfected with PCMV-CUA/UUA/CUA and pcDNA3.1-GFP-39TAG/TAAA/TGA cross-corresponding.
  • tRNA Pyl /PylRS has an efficient read-through effect on the perfect pairing stop codon, in which the read-through efficiency UAG is the highest, UGA is second, and UAA is the lowest.
  • the gene codon extension technique is applied to restore expression of a non-sense mutant protein associated with a human hereditary disease.
  • point mutation was performed at the corresponding position of the wild-type Dp71b sequence to construct Dp71b protein particle Dp71b 3116TAG (c.9346C>T) containing the premature stop codon UAG, Dp71b 3317TAG (c.9952C>T ), Dp71b 3601TAG (c.10801C>T).
  • the plasmid was transfected into the stable cell line HEK293-PYL, and the protein was extracted by adding non-natural amino acid for 48 hours.
  • the full-length protein of Dp71b was detected by western blot, and the expression of disease protein was restored.
  • the tRNA Pyl CUA / PylRS is used to verify endogenous premature stop codons at different positions with the stable cell line HEK293-PYL.
  • the Smad gene consisting of an intron and an exon was cloned into the pcDNA3 plasmid, and then the 39th, 122nd and 133th amino acid codons of Smad were mutated to the UAG premature stop codon by point mutation.
  • pcDNA3-Smad-39TAG, pcDNA3-Smad-122TAG and pcDNA3-Smad-133TAG are examples of Smad gene consisting of an intron and an exon.
  • the gene codon extension technique is used to read the nonsense mutation site of the tumor suppressor gene in the tumor cell.
  • the upper PCMV-CUA tRNA Pyl CUA / PylRS
  • Proteins were extracted 48 hours after the addition of unnatural amino acids, and the expression of full-length STK11 protein and full-length EPHB2 protein was restored in the tumor cell lines A549 and DU145 by western blot.
  • the present invention provides
  • tRNAPyl tRNA derived from M. oxysporum
  • PylRS pyrrolysyl-tRNA synthetase
  • Lys-diazirine NAEK
  • Lys-azido Shown Lys-diazirine (NAEK), Lys-azido shown, or
  • tRNA (tRNAPyl) of M. oxysporum as described in item 2 engineered from the anti-codon loop site-directed mutagenesis of the original tRNA Pyl CUA to tRNA Pyl UUA and tRNA Pyl UCA , the sequences corresponding to SEQ ID NO: 1 and SEQ ID, respectively. NO: 2, which is perfectly paired with the stop codons UAA and UGA, respectively.
  • tRNA Pyl UUA and tRNA Pyl UCA were constructed on PCMV-UUA and PCMV-UCA plasmid.
  • the direction from R 1 to R 2 is the N-terminus to the C-terminal direction of the amino acid sequence, and the N-th position may be any amino acid on the pathogenic protein or the tumor suppressor gene protein, and correspondingly, R 1 is the first to An amino acid residue at position N-1, and R 2 is an amino acid residue from the N+1th to the C-terminus,
  • R 1 to R 2 is the N-terminal to C-terminal direction of the amino acid sequence
  • the N-th position may be any one of the pathogenic protein or the tumor suppressor gene protein according to claim 1, correspondingly, R 1 is an amino acid residue at positions 1 to N-1, and R 2 is an amino acid residue at the N+1th to C-terminus,
  • the mutant plasmid was transfected into the stable cell line HEK293-PYL.
  • the protein was extracted after 48 hours of non-natural amino acid culture, and the full-length Smad protein was detected by western blot.
  • tRNAPyl CUA / PylRS, tRNAPyl UUA / PylRS and tRNAPyl UCA / PylRS read through the GFP green fluorescent protein TAG, TAA and TGA stop codons, respectively.
  • Figure 2A Establishment of a stable cell line HEK293-PYL screening method for orthogonal tRNA/aminoacyl tRNA synthetase.
  • Figure 2B Construction of a double virus overexpression system.
  • Figure 2C Construction of the pXH-12t-zeo vector.
  • Figure 3 Western blot verification of the protein expression in the stable cell line HEK293-PYL by reading the premature stop codon on the disease protein dystrophin.
  • Figure 5 Gene codon extension technology. Read the premature stop codon in A549 and DU145 tumor cell lines to restore STK11 and EPHB2 protein expression.
  • Example 1 PCMV-UUA (tRNA Pyl UUA / PylRS) and PCMV-UCA Construction of (tRNA Pyl UCA /PylRS) plasmid
  • PCMV General Microbial Culture Collection Management Center Culture Collection Address: Address: No. 1 Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, the deposit date is June 14, 2011, the deposit number is CGMCC No:4951 is classified as Escherichia coli, and plasmid pACYC-tRNA/PylRS is obtained from Escherichia coli pACYC-tRNA/PylRS containing plasmid pACYC-tRNA/PylRS (hereinafter referred to as PCMV).
  • PCMV General Microbial Culture Collection Management Center Culture Collection Address: Address: No. 1 Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, the deposit date is June 14, 2011, the deposit number is CGMCC No:4951 is classified as Escherichia coli, and plasmid pACYC-tRNA/PylRS is obtained from Escherichia coli pACYC-tRNA/
  • this plasmid can express tRNA synthetase (PylRS) which specifically recognizes the unnatural amino acids Lys-diazirine and Lys-azido and tRNA (tRNA Pyl CUA ) which specifically recognizes the amber stop codon UAG.
  • tRNA synthetase PylRS
  • tRNA Pyl CUA tRNA Pyl CUA
  • the inventors designed mutant primers for the anti-codon loop of the mutant tRNA Pyl CUA , and the specific primers are shown below.
  • PCMV-CUA as a template plasmid, using a site-directed mutagenesis kit ( Lightning Site-Directed Mutagenesis Kits, Catalog # 210518), according to instructions to tRNA Pyl CUA anti bases on the subring password CUA for point mutations by the above primer UUA and UCA, obtained PCMV-UUA (tRNA Pyl UUA / PylRS) and PCMV-UCA (tRNA Pyl UCA / PylRS) plasmids, mutagenesis was verified by sequencing.
  • the sequence of tRNA Pyl UUA is shown in SEQ ID NO: 1; the sequence of tRNA Pyl UCA is shown in SEQ ID NO: 2.
  • Example 2 Detection of GFP green fluorescent protein using a premature stop codon Read-through efficiency of tRNAPylCUA/UUA/UCA/PylRS orthogonal systems
  • the above product 2 was dissolved in pyridine, and 11 g of TsCl was added with stirring at 0 ° C overnight. After the reaction was completed, the reaction mixture was poured into a mixture of concentrated hydrochloric acid and ice water, and extracted with diethyl ether. The ether layer was washed with 1N hydrochloric acid and 1N NaOH. The organic phase dried column gave 11.8 g of a colorless viscous liquid product.
  • Green fluorescent protein GFP is the most commonly used reporter gene and a powerful tool for the insertion of non-natural amino acids, consisting of 238 amino acids, the gene sequence of which is SEQ ID NO: 7.
  • the GFP sequence was inserted into the pcDNA3.1 commercial plasmid, and the amino acid codon at position 39 of the GFP fluorescent gene was mutated to three premature stop codons of UAG, UAA and UGA, respectively. Primers capable of mutating the codons encoding the amino acids into three stop codons, respectively, are designed, the specific primers are shown in the following table.
  • the wild type GFP expression vector pcDNA3.1-GFP-WT was used as a template to mutate the amino acid codon at position 39 into three stop codons, respectively.
  • the expression plasmids (pcDNA3.1-GFP-39TAG, pcDNA3.1-GFP-39TAA and pcDNA3.1-GFP-39TGA) were sequenced and verified to be successful.
  • the pcDNA3.1-GFP obtained in the step 2 of Example 2, and the PCMV plasmid of the step 2 in Example 1 were mixed in a ratio of 1:2 according to the grouping of Table 3, and then the ratio of the transfection reagent megatrans 1.0 was 1:3.
  • 293T cells were added together.
  • NAEK was added to a concentration of 1 mM, and the cells were further cultured in an incubator at 37 ° C, 5% CO 2 for 48 hours, and then observed by fluorescence microscopy.
  • the results are shown in Figure 1. Show. It was finally confirmed that tRNAPyl/PylRS has an efficient read-through effect on the perfect pairing stop codon, in which the read-through efficiency UAG is the highest, UGA is second, and UAA is the lowest.
  • PCMV-TAG and pcDNA3.1-GFP-39TAG 2 PCMV-TAA and pcDNA3.1-GFP-39TAG 3 PCMV-TGA and pcDNA3.1-GFP-39TAG 4 PCMV-TAG and pcDNA3.1-GFP-39TAA 5 PCMV-TAA and pcDNA3.1-GFP-39TAA 6 PCMV-TGA and pcDNA3.1-GFP-39TAA 7 PCMV-TAG and pcDNA3.1-GFP-39TGA 8 PCMV-TAA and pcDNA3.1-GFP-39TGA 9 PCMV-TGA and pcDNA3.1-GFP-39TGA
  • Example 3 Reading the disease protein Dystrophin in three stable cell lines HEK293-PYL
  • Two lentiviral overexpression vectors with puromycin and hygromycin resistance were constructed, respectively carrying an aminoacyl tRNA synthetase and a reporter gene GFP with 39 TAG mutations, transducing HEK-293T cells by two rounds of virus Screened with puromycin/hygromycin to obtain a stable cell line PylRS/GFP 39TAG . Then, three pXH-zeo-12tRNA vectors carrying 12 copy number tRNA (CUA ⁇ UUA ⁇ UCA) and zeomycin resistance were constructed. The plasmid was linearized and transfected into PylRS/GFP 39TAG , and then screened by zeomycin in the presence of UAA.
  • GFP-positive cells were isolated (the cells were green in the presence of UAA, and the UAA cells were colorless), resulting in three stable cells expressing tRNA Pyl CUA /PylRS, tRNA Pyl UUA /PylRS and tRNA Pyl UCA /PylRS, respectively.
  • HEK293-PYL Fig. 2A.
  • an IRES sequence is inserted after the gene of interest, followed by a selectable marker gene, such that the transcribed mRNA can simultaneously express both proteins.
  • the use of the IRES system to overexpress the gene of interest has two advantages: 1. The target gene shares a promoter with the marker gene, avoiding the occurrence of false positives; 2. The efficiency of IRES translation is lower than that of the traditional translation initiation site, resulting in the expression of the target gene. Higher than the marker gene. Therefore, we introduced the CMV-pylRS sequence and the CMV-GFP 39TAG sequence in front of the IRES site by the BamHI restriction site, and obtained the double-virus system psd31-CMV-pylRS-IRES which can simultaneously overexpress two target proteins. -puro R /psd31-CMV-GFP39TAG-IRES-hygro R . The main primers used are shown in Table 4.
  • the inventors overexpressed tRNA by means of plasmid stable transfection.
  • the inventors constructed the vector pXH-12t-zeo, the sequence of which is shown in SEQ ID NO: 8. (Fig. 2C)
  • the psd31-CMV-pylRS-IRES-puro R virus was first packaged and transfected into HEK293T cells.
  • the concentration of puromycin was 0.6ug/ml.
  • psd31-CMV-GFP39TAG-IRES-hygro R virus was added.
  • the hygromycin screening concentration was 200 ug/ml, and the stable cell line 2 was obtained.
  • the inventors performed a third round of screening by stable plasmid transfection, and finally obtained a special cell line stably expressing orthogonal tRNA/aminoacyl tRNA synthetase.
  • the steps are as follows:
  • the stable cell line No. 2 (10 cm culture dish, 10 ug of plasmid per dish, transfected with no antibiotics) was transfected with pylRS and GFP 39TAG protein.
  • the GFP-positive clones were isolated and purified, and the culture was further expanded with a dose-reduced zeomycin to obtain a 12t-zeo stable cell line HEK293-PYL.
  • the plasmid is stably transfected with cell density.
  • the cell density is thin, it is easy to die and form a clone.
  • a special culture solution can be used: the old culture solution at a cell confluence of 80% is sterilized by a filter, and the fresh culture solution is mixed at a ratio of 1:1 for use. Or increase the serum concentration as appropriate.
  • the Dp71b sequence of the Dystrophin protein is shown in SEQ ID NO: 9, and the inventors performed point mutations on the wild-type Dp71b sequence according to the site of nonsense mutation in Duchenne muscular dystrophy patients, and introduced an early termination password at different positions.
  • Dp71b plasmid Dp71b 3116TAG c.9346C>T
  • Dp71b 3317TAG c.9952C>T
  • Dp71b 3601TAG c.10801C>T
  • the mutation was verified by sequencing.
  • the Dp71b 3116TAG , Dp71b 3317TAG , Dp71b 3601TAG plasmid obtained in the second step of Example 3 was mixed with the transfection reagent megatrans1.0 in a ratio of 1:3, and added to the stable cell line HEK293-PYL. After 6 hours, the concentration of the solution was changed to 1 mM NAEK, the cells were cultured in an incubator at 37 ° C, 5% CO 2 for 48 hours, and the protein was extracted by western blot (the primary antibody was anti-dystrophin, the anti-dystrophin protein C-terminal antibody, catalog number 12715-1-AP ) to the production of full-length dystrophin protein, as shown in Figure 3. It is proved that tRNA Pyl / PylRS can read the early stop codons at different positions to restore the expression of disease proteins.
  • Example 4 Read endogenous early termination password in stable cell line HEK293-PYL Sub-effect investigation
  • the Smad gene sequence consisting of an intron and an exon (as shown in SEQ ID: 3) was inserted into the pcDNA3.1 commercial plasmid, and then the 39th, 122 and 133 amino acid codons of Smad were mutated to UAG in advance.
  • the stop codon was used to obtain plasmids pcDNA3-Smad-39TAG, pcDNA3-Smad-122TAG and pcDNA3-Smad-133TAG (as shown in SEQ IDs: 4-6).
  • the pcDNA3-Smad-39TAG, pcDNA3-Smad-122TAG or pcDNA3-Smad-133TAG plasmid obtained with the first step of Example 4 and the transfection reagent Megatrans1.0 was mixed in a ratio of 1:3 and added to the stable cell line HEK293-PYL. After 6 hours, NAEK was added to the concentration of 1 mM, and the cells were further cultured in the incubator at 37 ° C, 5% CO 2 for 48 hours. Protein, western blot (primary antibody anti-myc, tag antibody) to full-length Smad protein production, as shown in Figure 4.
  • the verification gene codon extension technology can inhibit the non-sense-mediated mRNA degradation process and read the premature stop codon to restore protein expression.
  • STK11 in human lung cancer cell A 549 genome has a nonsense mutation c.109C>T, p.Q37X, which is amber stop codon UAG; human prostate cancer cell DU 145 genome EHPB2 gene nonsense mutation c. 2167C>T, p.Q723X, is the amber stop codon UAG.
  • the PCMV-CUA tRNA Pyl CUA / PylRS plasmid transfection reagent according megatrans1.0 1:3 proportions were transfected DU 145 and A 549 cells, the medium was changed after 6 hours NAEK 1mM is added at a concentration to cells Proteins were extracted after incubation for 48 hours at 37 ° C in a 5% CO 2 incubator. Western blot analysis (primary antibodies anti-STK11 and anti-EPHB2, respectively) was performed to STK11 and EPHB2 of the full-length protein, as shown in Figure 5.
  • the validation gene codon extension technology reads through the premature stop codon on the endogenous genome and restores the expression of the tumor suppressor gene protein.

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Abstract

提供了利用基因密码扩展的非天然氨基酸系统，高效率通读单基因遗传病中致病基因的无义突变位点，恢复突变体蛋白的正常结构和功能的方法。通过改造古甲烷球菌的tRNA(tRNAPyl)，得到全新的高通读效率的UAA和UGA编码的非天然氨基酸系统，扩展了tRNAPyl和吡咯赖氨酰-tRNA合成酶(PylRS)正交对的使用范围。构建了模拟内源性提前终止密码子的质粒，用于评价通读内源性提前终止密码子的效率。还提供了主要包括单基因遗传病的致病基因和肿瘤细胞内的肿瘤抑制基因的系统。

Description

利用优化的基因密码子扩展系统通读提前终止密码子疾病中的截短蛋白 技术领域
本发明属于生物制药领域，具体涉及利用基因密码扩展的非天然氨基酸系统，通读单基因遗传病的无义突变位点。并且通过改造古甲烷球菌的tRNA(tRNAPyl)，得到全新的高通读效率的UAA和UGA编码的非天然氨基酸系统，扩展了tRNAPyl和吡咯赖氨酰-tRNA合成酶(PylRS)正交对的使用范围，可用于通读三种无义突变的终止密码子UAG，UAA和UGA。
背景技术
无义突变导致的遗传性疾病
人类基因组中的存在许多种基因突变类型，无义突变是基因突变中的一类。基因突变是基因组DNA分子发生的可遗传的变异现象，其中包括移码突变和碱基置换。移码突变包括碱基的插入和缺失，碱基置换主要为错义突变和无义突变。无义突变是指编码基因的某个碱基发生突变，产生终止密码子UAG、UAA和UGA，终止密码子不编码任何氨基酸。终止密码子不能与转移RNA(tRNA)的反密码子配对，但能被终止因子或释放因子识别，终止肽键的合成，使蛋白质合成终止，因而生成不完整和没有功能的蛋白质。无义突变的发生使基因框内产生提前终止密码子(Premature termination codons，PTC)，导致基因编码的两种结果，一种即产生截短型蛋白，另一种则导致含有PTC的mRNA的稳定性降低，从而引发无义介导的mRNA降解途径(NMD)。据统计，大约有11.2％的遗传性疾病会产生PTC突变，被称作提前终止密码子病(Premature termination codons diseases，PTC diseases)，另一方面，许多癌症发生也会产生PTC突变(KEELING K.M.，WANG D.，CONARD S.E.，BEDWELL D.M..Suppression of premature termination codons as a therapeutic approach.Critical reviews in biochemistry and molecular biology，2012，47：444-463.)。
杜氏肌营养不良(Duchennemuscular dystrophy，DMD)是PTC疾病中的一个典型代表。DMD是一种严重的肌肉萎缩疾病，也是最常见的X连锁隐性遗传性疾病，以进展性、致死性为主要特点。DMD基 因的无义突变则是导致DMD发生的主要原因之一。无义突变产生提前终止密码子UAG、UAA、UGA，生成截短了的多肽产物，使患者缺失或缺少功能性的抗萎缩蛋白(dystrophin)，致使肌肉萎缩。据报道，Duchenne型肌营养不良症在活产男婴中的发病率为1/6300～1/3500[Dooley J，Gordon KE，Dodds L，MacSween J.Duchenne muscular dystrophy：a 30-year population-based incidence study.Clin Pediatr(Phila)，2010，49：177-179.]。该病目前尚无有效治愈方法，多于幼年期发病，青少年期丧失行走能力，成年早期死亡，给患者个人、家庭和社会造成沉重心理和经济负担。
先前的研究中通读提前终止密码子的方法有：(1)化学小分子诱导的通读：氨基糖苷类药物如G418和非氨基糖苷类药物如PTC124。1996年，Howard等在对囊性纤维变性的研究过程中首次观察到氨基糖苷类抗生素可以在哺乳动物细胞内诱导PTC通读，从而合成完整的有功能的蛋白质。但氨基糖苷类抗生素在发挥无义抑制作用的同时，可产生严重的不良反应，其中最严重的是耳毒性和肾毒性。并且在2016年2月，PTC124刚被美国FDA拒绝受理(2)外显子跳跃方法：针对跳过外显子51表达DMD患者的反义核苷酸药物。但FDA已经拒绝了BioMarin的drisapersen。而另一家公司Sarepta Therapeutics的Eteplirsen在2016年5月将会得到FDA的评审结果；(3)抑制子tRNA通读：其反密码子环发生突变而能和终止密码子配对，以致能通读终止密码子。该治疗手段难以进入临床应用的主要原因抑制子tRNA可能会识别正常终止密码子从而导致异常蛋白的潜在毒性。
遗传密码扩展技术
经过数年的研究，人们对原核生物核糖体的翻译机制已有较全面的理解，多种核糖体不同功能状态的晶体和电镜结构已得到解析，大多数氨tRNA合成酶的结构也已获得。基于这些研究成果，近年来发展起来了遗传密码扩展的技术-利用琥珀终止密码子(TAG)来编码多种非天然氨基酸并在生物活体内将其定点插入。到目前为止，这一技术已经将数种非天然氨基酸成功地定点表达在活细胞的蛋白质当中，赋予了这些蛋白质新颖的物理、化学和生理性质。使用这一方法，可以将非天然氨基酸(包括亲和标记和光致异构化的氨基酸、羰基氨基酸和糖基化氨基酸)引入蛋白质中(L.Wang等人，(2001)，SCIENCE  292：498-500；J.W.Chin等，2002，Journal of the American Chemical Society 124：9026-9027；J.W.Chin，&P.G.Schultz，2002，ChemBioChem 11：1135-1137)。这些研究表明，有可能且有选择性且常规地引入化学官能基团到蛋白质中，例如，羰基、炔基、和叠氮基团等特殊化学基团，这些基团一般能够有效且选择性地形成稳定的共价键，引入致病蛋白后可以用于研究致病蛋白和其他蛋白相互作用的机制。
经观察tRNAPyl和PylRS合成酶的复合体的晶体结构，PylRS合成酶不识别tRNAPyl的反密码子环，所以我们认为改变tRNAPyl的反密码子环的碱基序列不影响tRNAPyl和PylRS合成酶的正交性。
非天然氨酰tRNA合成酶是来自古菌或大肠杆菌等微生物的氨酰tRNA合成酶，经过正负筛选人工蛋白序列改造，不与大肠杆菌或真核生物内源的tRNA结合的具有特定序列的氨酰tRNA合成酶。参考文献：Wang L，Schultz P G.Expanding the genetic code[J].Angewandte chemie international edition，2005，44(1)：34-66。
本文所使用的tRNA与非天然氨酰tRNA合成酶“正交”的含义意味着这种tRNA不是任何内源性氨酰tRNA合成酶的底物，这种氨酰tRNA合成酶不能够氨酰化任何内源性的tRNA。这种正交对互相之间存在唯一对应关系。关于正交的含义还可以参考文献：Wang L，Schultz P G.Expanding the genetic code[J].Angewandte chemie international edition，2005，44(1)：34-66。
发明内容
发明人经过对现有技术的思考和研究，通过改造古甲烷球菌的tRNA(tRNAPyl)，构建了PCMV-UUA(tRNAPyl UUA/PylRS)和PCMV-UCA(tRNAPyl UCA/PylRS)质粒，得到全新的高通读效率的UAA和UGA编码的非天然氨基酸系统，使之能够用于通读三种终止密码子UAG，UAA和UGA；构建了模拟内源性提前终止密码子的质粒——在由内含子和外显子组成的Smad基因上引入提前终止密码子，可以用于评价通读内源性提前终止密码子的效率；将基因密码子扩展技术用于通读单基因遗传病的和肿瘤细胞中抑癌基因的无义突变位点，恢复相应蛋白质的表达。
本发明的优点可体现在如下中的一个或几个：
1、获得了全新的高通读效率的UAA和UGA编码的非天然氨基酸系统。
2、利用基因密码子扩展技术，实现通读遗传性疾病中的无义突变，恢复截短蛋白的正常结构和功能。
在一方面，本发明涉及tRNA，其中所述tRNA反密码子环上的碱基CUA突变为UUA或UCA，并且突变后的tRNA还能被至少一种与其正交的非天然氨酰tRNA合成酶识别。
在一方面，本发明涉及tRNA，其中所述tRNA的反密码子环不与至少一种与其正交的非天然氨酰tRNA合成酶结合。
在一方面，本发明涉及tRNA，其中所述tRNA为源自古甲烷球菌的tRNA(tRNAPyl)，或源自大肠杆菌的tRNA，优选地，所述tRNA为tRNAPyl UUA，其序列为SEQ ID NO：1，或者tRNAPyl UCA，其序列为SEQ ID NO：2。
在一方面，本发明涉及非天然氨基酸系统，其中所述系统包括本发明任一方面的tRNA和与其正交的至少一种非天然氨酰tRNA合成酶或其编码核酸序列，优选地，所述非天然氨基酸系统选自赖氨酸类非天然氨基酸系统、亮氨酸类非天然氨基酸系统和酪氨酸类非天然氨基酸系统，任选地，其中所述赖氨酸类非天然氨基酸系统包括源自古甲烷球菌的tRNA(tRNAPyl)和吡咯赖氨酰-tRNA合成酶(PylRS)，所述亮氨酸类非天然氨基酸系统包括源自大肠杆菌的tRNA(tRNALeu)、亮氨酰-tRNA合成酶(LeuRS)，所述酪氨酸类非天然氨基酸系统包括源自大肠杆菌的tRNA(tRNATyr)、酪氨酰-tRNA合成酶(TyrRS)。
在一方面，本发明涉及非天然氨基酸系统，所述赖氨酸类非天然氨基酸选自：
Figure PCTCN2017075577-appb-000001
所示的Lys-diazirine(NAEK)，
Figure PCTCN2017075577-appb-000002
所示的Lys-azido，或
其它含有双吖丙啶、叠氮结构的非天然氨基酸中的至少1种，所 述亮氨酸类非天然氨基酸选自：
Figure PCTCN2017075577-appb-000003
所示的Anap，所述酪氨酸类非天然氨基酸选自：
Figure PCTCN2017075577-appb-000004
所示的pAcF。
在一方面，本发明涉及包含本发明任一方面的tRNA或本发明任一方面的非天然氨基酸系统的质粒、载体、宿主细胞或试剂盒。
在一方面，本发明涉及基因密码子扩展的方法，其中将tRNA反密码子环上的碱基CUA进行点突变为UUA和UCA，并且突变后的tRNA还能被与其对应的非天然氨基酸tRNA合成酶识别。
本发明任一方面的方法，其中所述tRNA是为源自古甲烷球菌的tRNA(tRNAPyl)，或源自大肠杆菌的tRNA，优选地，所述tRNA为tRNAPyl UUA，其序列为SEQ ID NO：1，或者tRNAPyl UCA，其序列为SEQ ID NO：2。
在一方面，本发明涉及本发明任一方面的tRNA或本发明任一方面的非天然氨基酸系统在制备用于治疗遗传病或癌症的药物中的用途，其中所述遗传病或癌症是由于基因无义突变引起，优选地，所述遗传病或癌症是Dystrophin蛋白、抑癌基因STK11或EPHB2蛋白中发生无义突变导致。
本发明任一方面的用途，其中所述遗传病和癌症选自：杜氏肌营养不良、囊肿性纤维化、血友病A、血友病B、脂质储积症、共济失调毛细血管扩张、赫勒氏综合症、家族黑蒙性白痴、胃癌、肺癌。
在一方面，本发明涉及通读恢复无义突变体蛋白的正常表达和功能的方法，其中将本发明任一方面的tRNA或本发明任一方面的非天然氨基酸系统导入含有无义突变体蛋白的细胞或生物体内。
本发明任一方面的方法，其中所导入的tRNA或非天然氨基酸系统识别目标蛋白的无义突变，并且在无义突变的相应位点引入非天然氨基酸以使得目标蛋白的翻译得以避免提前终止并合成完整的有功能的蛋白质。
本发明任一方面的方法，其中所引入的非天然氨基酸是位于第N位的Lys-diazirine，其在蛋白中的连接方式如下式所示：
Figure PCTCN2017075577-appb-000005
其中，由R1到R2的方向为氨基酸序列的N末端到C末端方向，第N位可以是致病蛋白或抑癌基因蛋白上的任意一位氨基酸，相应地，R1为第1至第N-1位氨基酸残基，R2为第N+1位至C末端的氨基酸残基，R3为
Figure PCTCN2017075577-appb-000006
或者
所引入的非天然氨基酸是位于第N位的Lys-azido，其在致病蛋白或抑癌基因蛋白中的连接方式如下式所示：
Figure PCTCN2017075577-appb-000007
其中，由R1到R2的方向为氨基酸序列的N末端到C末端方向，第N位可以是如权利要求1所述的致病蛋白或抑癌基因蛋白上的任意一位，相应地，R1为第1至第N-1位氨基酸残基，R2为第N+1位至C末端的氨基酸残基，
R4为
Figure PCTCN2017075577-appb-000008
在一方面，本发明涉及哺乳动物稳定细胞系，其为HEK293-PYL，保藏日为2015年11月17日、其保藏号为CGMCC No：11592。
评价基因密码子扩展技术的方法，其特征在于其通读效率是用内源性提终止密码子质粒表达Smad蛋白的量评价，优选通过以下步骤：
(1)将Smad基因克隆到pcDNA3上，优选Smad基因序列如SEQ ID NO：3所示；
(2)将39，122，133位密码子突变为UAG琥珀型终止密码子， 得到突变体质粒pcDNA3-Smad-39TAG，pcDNA3-Smad-122TAG和pcDNA3-Smad-133TAG，优选所述突变体序列如SEQ ID NO：4-6所示；
(3)用突变体质粒转染稳定细胞系HEK293-PYL，加入非天然氨基酸培养1-96小时，优选12-72小时，最优选48小时后提取蛋白，经western blot检测到全长Smad蛋白，根据全长Smad蛋白表达量的多少评价通读效率。
在一方面，本发明涉及引物对，其中，所述引物对序列为PCMV-UAG-UAA-for：TGTAGATCGAATGGACTTTAAATCCGTTCAGCCGG和PCMV-UAG-UAA-rev：CCGGCTGAACGGATTTAAAGTCCATTCGATCTACA
或
PCMV-UAG-UGA-for：CATGTAGATCGAATGGACTTCAAATCCGTTCAGCCGGGTT和PCMV-UAG-UGA-rev：AACCCGGCTGAACGGATTTGAAGTCCATTCGATCTACATG。
在一方面，本发明涉及利用优化的基因密码子扩展技术，在无义突变体蛋白的提前终止密码子处插入非天然氨基酸来，通读恢复单基因遗传病中的致病蛋白和肿瘤细胞内抑癌基因蛋白的正常表达和功能的方法。
在一方面，本发明涉及古甲烷球菌的tRNA(tRNAPyl)，由原始tRNAPyl CUA的反密码子环定点突变改造成tRNAPyl UUA和tRNAPyl UCA，序列分别对应SEQ ID NO：1和SEQ ID NO：2，其特征在于分别与终止密码子UAA、UGA完美配对。tRNAPyl UUA和tRNAPyl UCA分别构建在PCMV-UUA和PCMV-UCA质粒上。
在一方面，本发明涉及致病蛋白或抑癌基因蛋白，其中所插入的非天然氨基酸是位于第N位的Lys-diazirine，其在蛋白中的连接方式如下式所示：
Figure PCTCN2017075577-appb-000009
其中，由R1到R2的方向为氨基酸序列的N末端到C末端方向，第N位可以是致病蛋白或抑癌基因蛋白上的任意一位氨基酸，相应地，R1为第1至第N-1位氨基酸残基，R2为第N+1位至C末端的氨基酸残基，
R3为
Figure PCTCN2017075577-appb-000010
在一方面，本发明涉及致病蛋白或抑癌基因蛋白，其中所引入的非天然氨基酸是位于第N位的Lys-azido，其在致病蛋白或抑癌基因蛋白中的连接方式如下式所示：
Figure PCTCN2017075577-appb-000011
其中，由R1到R2的方向为氨基酸序列的N末端到C末端方向，第N位可以是如权利要求1所述的致病蛋白或抑癌基因蛋白上的任意一位，相应地，R1为第1至第N-1位氨基酸残基，R2为第N+1位至C末端的氨基酸残基，
R4为
Figure PCTCN2017075577-appb-000012
在一方面，本发明涉及基因密码子扩展技术，其通读效率是用内源性提终止密码子质粒pcDNA3-Smad表达Smad蛋白的量评价。通过以下步骤：
(1)将原序列如SEQ ID NO：3所示的Smad基因克隆到pcDNA3上。
(2)将39，122，133位密码子突变为UAG琥珀型终止密码子，突变体质粒pcDNA3-Smad-39TAG，pcDNA3-Smad-122TAG和 pcDNA3-Smad-133TAG。序列如SEQ ID NO：4～6所示。
(3)突变体质粒转染稳定细胞系HEK293-PYL，加入非天然氨基酸培养48小时后提取蛋白，经western blot检测到全长Smad蛋白。
在一方面，本发明涉及可稳定表达tRNA(tRNAPyl CUA)和吡咯赖氨酰-tRNA合成酶(PylRS)的哺乳动物稳定细胞系，其为HEK293-PYL，保藏日为2015年11月17日、其保藏号为CGMCC No：11592。以及稳定整合表达tRNAPyl UUA/PylRS的HEK293-PYL-TAA稳定细胞系，和稳定表达tRNAPyl UCA/PylRS的HEK293-PYL-TGA稳定细胞系。
具体实施方案
具体地，在本发明的一个具体的实施方案中，构建了识别三种终止密码子(琥珀型(amber)、赭石型(ocher)、乳白型(opal))的三种tRNAPyl/PylRS质粒，并在稳定细胞系HEK293-PYL(已保藏于中国普通微生物菌种保藏管理中心，保藏日为2015年11月17日、保藏号为CGMCC No：11592。其分类命名为人HEK293T细胞)中恢复DMD疾病蛋白Dystrophin的表达、通读了内源性提前终止密码子和在A549和DU145肿瘤细胞系中恢复了抑癌基因STK11和EPHB2蛋白的表达。主要通过以下6个步骤：(1)构建PCMV-UUA(tRNAPyl UUA/PylRS)和PCMV-UCA(tRNAPyl UCA/PylRS)质粒；(2)构建含有提前终止密码子的GFP报告基因pcDNA3.1-GFP-39TAG；pcDNA3.1-GFP-39TAA；pcDNA3.1-GFP-39TGA；(3)根据DMD病人的无义突变位点，利用点突变技术在dystrophin蛋白的同源异构体蛋白Dp71b蛋白相应位点引入提前终止密码子，构建含有提前终止密码子UAG的Dp71b蛋白质粒Dp71b3116TAG，Dp71b3317TAG，Dp71b3601TAG；(4)在Smad基因(由内含子和外显子组成)上引入提前终止密码子TAG，构建了模拟内源性提前终止密码子的质粒pcDNA3-Smad-39TAG，pcDNA3-Smad-122TAG和pcDNA3-Smad-133TAG；(5)将步骤(1)和(2)的质粒交叉对应转染至293T细胞，加入非天然氨基酸培养48小时后观察绿色荧光，比较三种终止密码子的通读效率；(6)将步骤(3)中的质粒转染稳定细胞系HEK293-PYL，加入非天然氨基酸培养48小时后提蛋白，western blot方法检测到Dp71b全长蛋白，疾病蛋白表达恢复；(7)将步骤(4)中的质粒转染稳定细胞系HEK293-PYL，加入非天 然氨基酸培养48小时后提蛋白，western blot方法检测Smad全长蛋白，证明基因密码子扩展技术能够有效抑制无义介导的mRNA降解途径，通读不同位置内源性的提前终止密码子；(8)将PCMV-CUA(tRNAPyl CUA/PylRS)转染肿瘤细胞系A549和DU145，加入非天然氨基酸培养48小时后提取蛋白，经western blot证明在肿瘤细胞系A549和DU145中STK11蛋白和全长EPHB2蛋白恢复表达。
在本发明的一个具体的实施方案中，以PCMV-CUA(tRNAPyl CUA/PylRS)作为模板质粒，设计点突变引物，利用定点突变试剂盒，将tRNAPyl CUA反密码子环上的碱基CUA通过上述引物进行点突变为UUA和UCA，获得PCMV-UUA(tRNAPyl UUA/PylRS)和PCMV-UCA(tRNAPyl UCA/PylRS)质粒。
在本发明的一个具体的实施方案中，利用含有提前终止密码子的GFP绿色荧光蛋白检测三种tRNAPyl CUA/UUA/UCA/PylRS的通读效率。第一步利用点突变技术将GFP荧光基因第39位氨基酸密码子分别点突变为UAG、UAA、UGA三种提前终止密码子得到pcDNA3.1-GFP-39TAG、pcDNA3.1-GFP-39TAA和pcDNA3.1-GFP-39TGA三个质粒。第二步用PCMV-CUA/UUA/CUA和pcDNA3.1-GFP-39TAG/TAAA/TGA交叉对应共转染293T细胞。第三步加入非天然氨基酸培养48小时后用荧光显微镜观察绿色荧光。最终确认tRNAPyl/PylRS对与之完美配对的终止密码子有高效的通读作用，其中通读效率UAG最高，UGA次之，UAA最低。
在本发明的一个具体的实施方案中，将基因密码子扩展技术应用于恢复人类遗传性疾病相关无义突变蛋白表达。根据人类DMD疾病中无义突变位置，在野生型Dp71b序列对应位置进行点突变，构建含有提前终止密码子UAG的Dp71b蛋白质粒Dp71b3116TAG(c.9346C＞T)，Dp71b3317TAG(c.9952C＞T)，Dp71b3601TAG(c.10801C＞T)。将质粒转染稳定细胞系HEK293-PYL，加入非天然氨基酸培养48小时后提蛋白，western blot方法检测到Dp71b全长蛋白，疾病蛋白表达恢复。
在本发明的一个具体的实施方案中，用稳定细胞系HEK293-PYL验证tRNAPyl CUA/PylRS通读不同位置的内源性提前终止密码子。第一步将由内含子和外显子组成的Smad基因克隆到pcDNA3质粒上，然后利用点突变方法将Smad第39位，122位和133位氨基酸密码子突变 为UAG提前终止密码子，得到质粒pcDNA3-Smad-39TAG，pcDNA3-Smad-122TAG和pcDNA3-Smad-133TAG。将内源性提前终止密码子质粒(pcDNA3-Smad-39TAG，pcDNA3-Smad-122TAG或pcDNA3-Smad-133TAG)顺时转染含有古甲烷球菌的tRNA(tRNAPyl)和吡咯赖氨酰-tRNA合成酶(PylRS)的稳定细胞系。加入非天然氨基酸后培养48小时后提取蛋白，经western blot三组都检测到全长Smad的蛋白。证明基因密码子扩展技术能够有效抑制无义介导的mRNA降解途径，通读不同位置内源性的提前终止密码子，恢复全长蛋白的表达。
在本发明的一个具体的实施方案中，将基因密码子扩展技术用完通读肿瘤细胞中抑癌基因的无义突变位点。将PCMV-CUA(tRNAPyl CUA/PylRS)转染肿瘤细胞系A549和DU145(人肺癌细胞A 549基因组上STK11发生无义突变c.109C＞T，p.Q37X，为终止密码子UAG；人前列腺癌细胞DU 145基因组上EPHB2基因发生无义突变c.2167C＞T，p.Q723X，为终止密码子UAG)。加入非天然氨基酸培养48小时后提取蛋白，经western blot证明基因密码子扩展技术在肿瘤细胞系A549和DU145中恢复了全长STK11蛋白和全长EPHB2蛋白的表达。
更为具体地，本发明提供了
1.利用基因密码子扩展技术，在无义突变体蛋白的提前终止密码子处插入非天然氨基酸来，通读恢复单基因遗传病中的致病蛋白和肿瘤细胞内抑癌基因蛋白的正常表达和功能的方法。
2.如项目1所述的基因密码子扩展技术，由源自古甲烷球菌的tRNA(tRNAPyl)、吡咯赖氨酰-tRNA合成酶(PylRS)和非天然氨基酸组成。所述非天然氨基酸选自：
Figure PCTCN2017075577-appb-000013
所示的Lys-diazirine(NAEK)，
Figure PCTCN2017075577-appb-000014
所示的Lys-azido，或
其它含有双吖丙啶、叠氮结构的非天然氨基酸中的至少1种。
3.如项目2所述的古甲烷球菌的tRNA(tRNAPyl)，由原始tRNAPyl CUA的反密码子环定点突变改造成tRNAPyl UUA和tRNAPyl UCA，序列分别对应SEQ ID NO：1和SEQ ID NO：2，其中分别与终止密码子UAA、UGA完美配对。tRNAPyl UUA和tRNAPyl UCA分别构建在PCMV-UUA和PCMV-UCA质粒上。
4.如项目1所述的致病蛋白或抑癌基因蛋白，其中所插入的非天然氨基酸是位于第N位的Lys-diazirine，其在蛋白中的连接方式如下式所示：
5.
Figure PCTCN2017075577-appb-000015
其中，由R1到R2的方向为氨基酸序列的N末端到C末端方向，第N位可以是致病蛋白或抑癌基因蛋白上的任意一位氨基酸，相应地，R1为第1至第N-1位氨基酸残基，R2为第N+1位至C末端的氨基酸残基，
R3为
Figure PCTCN2017075577-appb-000016
6.如项目1所述的致病蛋白或抑癌基因蛋白，其中所引入的非天然氨基酸是位于第N位的Lys-azido，其在流感病毒蛋白中的连接方式如下式所示：
Figure PCTCN2017075577-appb-000017
其中，由R1到R2的方向为氨基酸序列的N末端到C末端方向，第N位可以是如权利要求1所述的致病蛋白或抑癌基因蛋白上的任意一位，相应地，R1为第1至第N-1位氨基酸残基，R2为第N+1位至C末端的氨基酸残基，
R4为
Figure PCTCN2017075577-appb-000018
7.如项目1-5所述的基因密码子扩展技术，其通读效率是用内源性提终止密码子质粒pcDNA3-Smad表达Smad蛋白的量评价。通过以下步骤：
(1)将原序列如SEQ ID NO：3所示的Smad基因克隆到pcDNA3上。
(2)将39，122，133位密码子突变为UAG琥珀型终止密码子，突变体质粒pcDNA3-Smad-39TAG，pcDNA3-Smad-122TAG和pcDNA3-Smad-133TAG。序列如SEQ ID NO：4～6所示。
(3)突变体质粒转染稳定细胞系HEK293-PYL，加入非天然氨基酸培养48小时后提取蛋白，经western blot检测到全长Smad蛋白。
8.可稳定表达tRNA(tRNAPyl)和吡咯赖氨酰-tRNA合成酶(tRNAPyl)的哺乳动物稳定细胞系，其为HEK293-PYL，保藏日为2015年11月17日、其保藏号为CGMCC No：11592。
附图说明：
图1.tRNAPylCUA/PylRS，tRNAPylUUA/PylRS和tRNAPylUCA/PylRS分别通读GFP绿色荧光蛋白TAG，TAA和TGA终止密码子。
图2A.正交tRNA/氨酰tRNA合成酶的稳定细胞系HEK293-PYL筛选方法的建立。
图2B.双病毒过表达体系的构建。
图2C.pXH-12t-zeo载体的构建。
图3.Western blot验证在稳定细胞系HEK293-PYL中，通读疾病蛋白dystrophin上的提前终止密码子从而恢复蛋白表达。
图4.稳定细胞系HEK293-PYL中通读内源性提前终止密码子
图5.基因密码子扩展技术通读A549和DU145肿瘤细胞系中提提前终止密码子，恢复STK11和EPHB2蛋白表达
以上所述的仅是本发明的一些实施方式。对于本领域的普通技术人员来说，在不脱离本发明创造构思的前提下，还可以做出若干变形 和改进，这些都属于本发明的保护范围。
为了更好地理解本发明，发明人用实施例对具体试验进行阐述和说明，其中所述实施例仅用于说明，并不限定本发明的保护范围。任何与本发明等价的变体或者实施方案都包括在本发明中。
实施例1：PCMV-UUA(tRNAPyl UUA/PylRS)和PCMV-UCA (tRNAPyl UCA/PylRS)质粒的构建
(1)古甲烷球菌PCMV-CUA质粒(tRNAPyl CUA/PylRS)的获得
从保藏地：中国普通微生物菌种保藏管理中心菌种保藏地址：地址：北京市朝阳区北辰西路1号院，中国科学院微生物研究所，保藏日为2011年6月14日、保藏号为CGMCC No：4951的分类命名为大肠埃希氏菌(Escherichia coli)的含有质粒pACYC-tRNA/PylRS的大肠埃希氏菌pACYC-tRNA/PylRS中获取质粒pACYC-tRNA/PylRS(以下简称该质粒为PCMV-CUA)，该质粒可以表达特异识别非天然氨基酸Lys-diazirine和Lys-azido的tRNA合成酶(PylRS)和特异性识别琥珀终止密码子UAG的tRNA(tRNAPyl CUA)。
(2)点突变tRNAPyl CUA构建PCMV-UUA(tRNAPylUUA/PylRS)和PCMV-UCA(tRNAPylUCA/PylRS)质粒
发明人针对突变tRNAPyl CUA的反密码子环，分别设计突变引物，具体的引物如下所示。
表1 tRNAPyl CUA的反密码子环点突变引物
Figure PCTCN2017075577-appb-000019
以PCMV-CUA作为模板质粒，利用定点突变试剂盒(
Figure PCTCN2017075577-appb-000020
Lightning Site-Directed Mutagenesis Kits，Catalog#210518)，按说明书操作将tRNAPyl CUA反密码子环上的碱基CUA通过上述引物进行点突变为UUA和UCA，获得PCMV-UUA(tRNAPyl UUA/PylRS)和PCMV-UCA (tRNAPyl UCA/PylRS)质粒，经测序验证突变成功。tRNAPyl UUA的序列为SEQ ID NO：1所示；tRNAPyl UCA的序列为SEQ ID NO：2所示。
实施例2：利用含有提前终止密码子的GFP绿色荧光蛋白检测三 种tRNAPylCUA/UUA/UCA/PylRS正交系统的通读效率
(1)非天然氨基酸Lys-diazirine的合成和鉴定
非天然氨基酸Lys-diazirine的化学合成反应式如下。
Figure PCTCN2017075577-appb-000021
如上式所示，将原料1(5-羟基-2-戊酮)15mL与液氨40mL在-40℃下搅拌反应5h，之后降温至-60℃，缓慢滴加NH2OSO3H的甲醇溶液，加毕升至室温，反应过夜。滤除沉淀，向上清液中加入三乙胺，冰浴条件下缓慢加入I2，至反应液颜色变深，不再产生气泡为止。反应完全后蒸除溶剂，经乙醚萃取后干燥。蒸除乙醚，剩余液体减压蒸馏获得25.4g无色粘稠液体产物2。
将上述产物2用吡啶溶解，0℃搅拌下加入11g TsCl，反应过夜。待反应完全后将反应液倒入浓盐酸与冰水的混合液中，乙醚萃取，醚层分别用1N盐酸和1N NaOH洗涤。有机相干燥柱分得到11.8g无色粘稠液体产物3。
将上述产物3用DMF溶解，加入NaN3室温反应隔夜至反应完全，加入大量水，乙醚萃取。蒸除乙醚，剩余产物用THF∶水(9∶1)混溶，加入三苯基磷，室温反应。反应完后加1N HCl混匀，旋干THF，二氯甲 烷把未反应的原料，PPh3和O＝PPh3洗掉，液相加1N NaOH调pH到12，二氯甲烷萃取出4.0g产物4。
将5.2g原料5(Boc-Lys-OMe)与羰基二咪唑反应，制备出5.9g化合物6。之后化合物6与上述产物4(4.0g)偶联得到化合物7，最后经过两步脱保护，将Boc和甲酯脱除，得到目标4.5g产物8，即Lys-diazirine。经谱学验证，结果为：
1H NMR(400MHz，D2O)：δ3.10(1H，t，J＝6.3Hz)，2.96(4H，m)，1.25(10H，m)，0.90(3H，s)；13C NMR(100MHz，D2O)：183.63，160.66，56.00，39.80，39.30，34.49，30.84，29.20，26.75，23.92，22.43，18.80；HREIMS m/z 308.16937[M+1]+(calcd for C12H22N5NaO3，308.16931)，证明所得到的Lys-diazirine结构正确。
(2)构建含有提前终止密码子的GFP报告基因
绿色荧光蛋白GFP是最常用的报告基因，也是指示非天然氨基酸插入的有力工具，其由238个氨基酸组成，其基因序列如SEQ ID NO：7。
将GFP序列插入pcDNA3.1商业质粒上，将GFP荧光基因第39位氨基酸密码子分别点突变为UAG、UAA、UGA三种提前终止密码子。设计能够使编码所述氨基酸的密码子分别突变为三种终止密码子的引物，具体引物如下表所示。
表2 GFP突变引物列表
Figure PCTCN2017075577-appb-000022
利用定点突变试剂盒(
Figure PCTCN2017075577-appb-000023
Lightning Site-Directed Mutagenesis Kits，Catalog#210518)，按说明书操作，以野生型GFP表达载体pcDNA3.1-GFP-WT为模板将第39位的氨基酸密码子分别突变为三种终止密码子，构建得到表达质粒(pcDNA3.1-GFP-39TAG、pcDNA3.1-GFP-39TAA和pcDNA3.1-GFP-39TGA)，经测序验证突变成功。
(3)293T细胞中顺转PCMV和pcDNA3.1-GFP质粒验证突变后的正交系统的通读效率
将实施例2的步骤2获得的pcDNA3.1-GFP，以及实施例1的步骤2的PCMV质粒按表3的分组以1∶2比例混合，再与转染试剂megatrans1.0按1∶3比例混合，共同加入293T细胞，6小时后换液加入浓度为1mM的NAEK，至细胞于37℃，5％CO2的孵箱中继续培养48小时后后用荧光显微镜观察绿色荧光，结果如图1所示。最终确认tRNAPyl/PylRS对与之完美配对的终止密码子有高效的通读作用，其中通读效率UAG最高，UGA次之，UAA最低。
表3 PCMV质粒和GFP质粒分组混合
组别 质粒
1 PCMV-TAG和pcDNA3.1-GFP-39TAG
2 PCMV-TAA和pcDNA3.1-GFP-39TAG
3 PCMV-TGA和pcDNA3.1-GFP-39TAG
4 PCMV-TAG和pcDNA3.1-GFP-39TAA
5 PCMV-TAA和pcDNA3.1-GFP-39TAA
6 PCMV-TGA和pcDNA3.1-GFP-39TAA
7 PCMV-TAG和pcDNA3.1-GFP-39TGA
8 PCMV-TAA和pcDNA3.1-GFP-39TGA
9 PCMV-TGA和pcDNA3.1-GFP-39TGA
实例3：三种稳定细胞系HEK293-PYL中通读疾病蛋白Dystrophin
(1)稳定细胞系HEK293-PYL的构建
构建了分别带有puromycin和hygromycin抗性的2个慢病毒过表达载体，两者分别携带氨酰tRNA合成酶和带有39位TAG突变的报告基因GFP，通过两轮病毒转导HEK-293T细胞和puromycin/hygromycin 筛选，得到稳定细胞株PylRS/GFP39TAG。之后，构建了三种携带12个拷贝数tRNA(CUA\UUA\UCA)和zeomycin抗性的pXH-zeo-12tRNA载体，质粒线性化转染细胞株PylRS/GFP39TAG，后经UAA存在下zeomycin筛选，最后分离出GFP阳性细胞(UAA存在下细胞呈绿色，去除UAA细胞呈无色)，从而得到了分别表达tRNAPyl CUA/PylRS、tRNAPyl UUA/PylRS和tRNAPyl UCA/PylRS的三株稳定细胞系HEK293-PYL(图2A)。
a.载体的构建
我们首先构建了分别带有puromycin和hygromycin抗性的2个慢病毒过表达载体，两者分别携带氨酰tRNA合成酶和带有39位TAG突变的报告基因GFP，见图2B。我们从psd31载体出发，我们先通过BamHI/xbal酶切位点将sv40-puroR基因分别替换成IRES-puroR和IRES-hygroR基因，这样就得到了2个不同抗性的病毒载体psd31-IRES-puroR和psd31-IRES-hygroR。其中，IRES为内部核糖体进入序列(internal ribosome entry site)，IRES序列常用于多顺反子基因表达。例如，在目的基因之后插入IRES序列，后面是选择标记基因，这样转录出来的mRNA就可以同时表达两种蛋白。利用IRES系统过表达目的基因有2个优势：1.目的基因与标记基因共用一个启动子，避免了假阳性的出现；2.IRES翻译效率低于传统翻译起始位点，使得目的基因表达量高于标记基因。所以，我们在IRES位点的前面通过BamHI酶切位点分别引入CMV-pylRS序列和CMV-GFP39TAG序列，就得到了能同时过表达两个目的蛋白的双病毒体系psd31-CMV-pylRS-IRES-puroR/psd31-CMV-GFP39TAG-IRES-hygroR。所用主要引物见表4。
表4.双病毒构建引物
Figure PCTCN2017075577-appb-000024
发明人用质粒稳定转染的方法过表达tRNA。为了保证tRNA的表达量，发明人构建得到了载体pXH-12t-zeo，其序列如SEQ ID NO：8所示。(图2C)
b.慢病毒的包装和转导
先包装psd31-CMV-pylRS-IRES-puroR病毒，转导HEK293T细胞，puromycin筛选浓度为0.6ug/ml，得到稳定细胞系1号后，再加入psd31-CMV-GFP39TAG-IRES-hygroR病毒，hygromycin筛选浓度为200ug/ml，得到稳定细胞系2号。
c.质粒的稳定转染
发明人通过质粒稳定转染，进行了第三轮筛选，最后得到了稳定表达正交tRNA/氨酰tRNA合成酶的特殊细胞系，步骤如下：
A.将pXH-12t-zeo载体酶切线性化后，转染表达pylRS和GFP39TAG 蛋白的稳定细胞系2号(10cm培养皿，每皿10ug质粒，转染时不能有抗生素的存在)。
B.转染6小时后换液，加入非天然氨基酸。
C.转染48小时后，观察绿色荧光，换液，加入400ug/ml的zeomycin。
D.每3天换液，直到blank组全部死亡，转染组形成克隆。
E.分离纯化GFP阳性克隆，继续用剂量减半的zeomycin扩大培养，得到12t-zeo稳定细胞系HEK293-PYL。
质粒稳定转染筛选单克隆的要点如下：
A.质粒稳定转染细胞密度很重要，筛选时细胞密度偏稀，易死亡难形成克隆。
B.从单克隆化时开始，就要加大营养，血清和生长因子。
C.单克隆接种到孔中细胞数目很少时，细胞之间的信号会变得很弱，也会导致阳性细胞的状态不佳甚至死亡。可以使用一种特殊的培养液：即细胞汇合度达到80％的时候的旧培养液通过滤器消毒，和新鲜的培养液按1∶1混合备用。或者适当增加血清浓度。
D.单克隆消化后，不要加zeomycin和UAA，等细胞贴壁后再加，避免细胞死亡。
(2)构建含有提前终止密码子UAG的Dp71b突变质粒
Dystrophin蛋白的同源异构体Dp71b序列如SEQ ID NO：9所示，发明人根据杜氏肌营养不良病人无义突变的位点，对野生型Dp71b序列进行点突变，在不同位置引入提前终止密码子，构建含有提前终止密码子UAG的Dp71b质粒Dp71b3116TAG(c.9346C＞T)，Dp71b3317TAG(c.9952C＞T)，Dp71b3601TAG(c.10801C＞T)，如序列SEQ ID NO：10～12所示，经测序验证突变成功。
表5：Dp71b点突变引物
Figure PCTCN2017075577-appb-000025
(3)稳定细胞系HEK293-PYL中通读疾病蛋白Dystrophin
将实施例3的步骤2获得的Dp71b3116TAG，Dp71b3317TAG，Dp71b3601TAG质粒与转染试剂megatrans1.0按1∶3比例混合，共同加入稳定细胞系HEK293-PYL中，6小时后换液加入浓度为1mM的NAEK，至细胞于37℃，5％CO2的孵箱中继续培养48小时后提取蛋白，western blot检测(一抗为anti-dystrophin，为抗dystrophin蛋白C末端抗体，货号12715-1-AP)到全长dystrophin蛋白的产生，如图3。证明tRNAPyl/PylRS能够通读不同位置的提前终止密码子，恢复疾病蛋白的表达。
实施例4：稳定细胞系HEK293-PYL中通读内源性提前终止密码 子效果考察
(1)内源性提前终止密码子质粒pcDNA3.1-Smad-39TAG；pcDNA3.1-Smad-39TAA；pcDNA3.1-Smad-39TGA构建
将有内含子和外显子组成的Smad基因序列(如SEQ ID：3所示)插入pcDNA3.1商业质粒上，然后将Smad第39位，122位和133位氨基酸密码子突变为UAG提前终止密码子，得到质粒pcDNA3-Smad-39TAG，pcDNA3-Smad-122TAG和pcDNA3-Smad-133TAG(如SEQ ID：4～6所示)。
(2)稳定细胞系中验证内源性提前终止密码子的通读
将实施例4的步骤1获得的pcDNA3-Smad-39TAG，pcDNA3-Smad-122TAG或pcDNA3-Smad-133TAG质粒与转染试剂 megatrans1.0按1∶3比例混合，加入稳定细胞系HEK293-PYL中，6小时后换液加入浓度为1mM的NAEK，至细胞于37℃，5％CO2的孵箱中继续培养48小时后提取蛋白，western blot检测(一抗为anti-myc，标签抗体)到全长Smad蛋白的产生，如图4。验证基因密码子扩展技术能够抑制无义介导的mRNA降解过程，并通读提前终止密码子，恢复蛋白的表达。
实施例5：基因密码子扩展通读肿瘤细胞系基因组上的提前终止密 码子
经查阅文献，人肺癌细胞A 549基因组上STK11发生无义突变c.109C＞T，p.Q37X，为琥珀型终止密码子UAG；人前列腺癌细胞DU 145基因组上EPHB2基因发生无义突变c.2167C＞T，p.Q723X，为琥珀型终止密码子UAG。
将PCMV-CUA(tRNAPyl CUA/PylRS)质粒与转染试剂megatrans1.0按1∶3比例混合，分别转染A 549和DU 145细胞，6小时后换液加入浓度为1mM的NAEK，至细胞于37℃，5％CO2的孵箱中继续培养48小时后提取蛋白，western blot检测(一抗分别为anti-STK11和anti-EPHB2)到全长蛋白的STK11和EPHB2产生，如图5。验证基因密码子扩展技术能够通读内源性基因组上的提前终止密码子，恢复抑癌基因蛋白表达。

Claims (16)

  1. tRNA，其特征在于所述tRNA反密码子环上的碱基CUA突变为UUA或UCA，并且突变后的tRNA还能被至少一种与其正交的非天然氨酰tRNA合成酶识别。
  2. 权利要求1的tRNA，其中所述tRNA的反密码子环不与至少一种与其正交的非天然氨酰tRNA合成酶结合。
  3. 权利要求1或2的tRNA，其特征在于所述tRNA为源自古甲烷球菌的tRNA(tRNAPyl)，或源自大肠杆菌的tRNA，优选地，所述tRNA为tRNAPyl UUA，其序列为SEQ ID NO：1，或者tRNAPyl UCA，其序列为SEQ ID NO：2。
  4. 非天然氨基酸系统，其特征在于所述系统包括权利要求1-3任一项的tRNA和与其正交的至少一种非天然氨酰tRNA合成酶或其编码核酸序列，优选地，所述非天然氨基酸系统选自赖氨酸类非天然氨基酸系统、亮氨酸类非天然氨基酸系统和酪氨酸类非天然氨基酸系统，任选地，其中所述赖氨酸类非天然氨基酸系统包括源自古甲烷球菌的tRNA(tRNAPyl)和吡咯赖氨酰-tRNA合成酶(PylRS)，所述亮氨酸类非天然氨基酸系统包括源自大肠杆菌的tRNA(tRNALeu)、亮氨酰-tRNA合成酶(LeuRS)，所述酪氨酸类非天然氨基酸系统包括源自大肠杆菌的tRNA(tRNATyr)、酪氨酰-tRNA合成酶(TyrRS)。
  5. 根据权利要求4的非天然氨基酸系统，所述赖氨酸类非天然氨基酸选自：
    Figure PCTCN2017075577-appb-100001
    所示的Lys-diazirine(NAEK)，
    Figure PCTCN2017075577-appb-100002
    所示的Lys-azido，或
    其它含有双吖丙啶、叠氮结构的非天然氨基酸中的至少1种，所述亮氨酸类非天然氨基酸选自：
    Figure PCTCN2017075577-appb-100003
    所示的Anap， 所述酪氨酸类非天然氨基酸选自：
    Figure PCTCN2017075577-appb-100004
    所示的pAcF。
  6. 包含权利要求1-3任一项的tRNA或权利要求4-5任一项的非天然氨基酸系统的质粒、载体、宿主细胞或试剂盒。
  7. 基因密码子扩展的方法，其特征在于将tRNA反密码子环上的碱基CUA进行点突变为UUA和UCA，并且突变后的tRNA还能被与其对应的非天然氨基酸tRNA合成酶识别。
  8. 根据权利要求7的方法，其中所述tRNA是为源自古甲烷球菌的tRNA(tRNAPyl)，或源自大肠杆菌的tRNA，优选地，所述tRNA为tRNAPyl UUA，其序列为SEQ ID NO：1，或者tRNAPyl UCA，其序列为SEQ ID NO：2。
  9. 权利要求1-3任一项的tRNA或权利要求4-6任一项的非天然氨基酸系统在制备用于治疗遗传病或癌症的药物中的用途，其中所述遗传病或癌症是由于基因无义突变引起，优选地，所述遗传病或癌症是Dystrophin蛋白、抑癌基因STK11或EPHB2蛋白中发生无义突变导致。
  10. 根据权利要求9的用途，其中所述遗传病和癌症选自：杜氏肌营养不良、囊肿性纤维化、血友病A、血友病B、脂质储积症、共济失调毛细血管扩张、赫勒氏综合症、家族黑蒙性白痴、胃癌、肺癌。
  11. 通读恢复无义突变体蛋白的正常表达和功能的方法，其特征在于将权利要求1-3任一项的tRNA或权利要求3-6任一项的非天然氨基酸系统导入含有无义突变体蛋白的细胞或生物体内。
  12. 根据权利要求11的方法，其中所导入的tRNA或非天然氨基酸系统识别目标蛋白的无义突变，并且在无义突变的相应位点引入非天然氨基酸以使得目标蛋白的翻译得以避免提前终止并合成完整的有功能的蛋白质。
  13. 根据权利要求12的方法，其中所引入的非天然氨基酸是位于第N位的Lys-diazirine，其在蛋白中的连接方式如下式所示：
    Figure PCTCN2017075577-appb-100005
    其中，由R1到R2的方向为氨基酸序列的N末端到C末端方向，第N位可以是致病蛋白或抑癌基因蛋白上的任意一位氨基酸，相应地，R1为第1至第N-1位氨基酸残基，R2为第N+1位至C末端的氨基酸残基，R3为
    Figure PCTCN2017075577-appb-100006
    或者
    所引入的非天然氨基酸是位于第N位的Lys-azido，其在致病蛋白或抑癌基因蛋白中的连接方式如下式所示：
    Figure PCTCN2017075577-appb-100007
    其中，由R1到R2的方向为氨基酸序列的N末端到C末端方向，第N位可以是如权利要求1所述的致病蛋白或抑癌基因蛋白上的任意一位，相应地，R1为第1至第N-1位氨基酸残基，R2为第N+1位至C末端的氨基酸残基，
    R4为
    Figure PCTCN2017075577-appb-100008
  14. 哺乳动物稳定细胞系，其为HEK293-PYL，保藏日为2015年11月17日、其保藏号为CGMCC No：11592。
  15. 评价基因密码子扩展技术的方法，其特征在于其通读效率是用内源性提终止密码子质粒表达Smad蛋白的量评价，优选通过以下步骤：
    (1)将Smad基因克隆到pcDNA3上，优选Smad基因序列如SEQ ID NO：3所示；
    (2)将39，122，133位密码子突变为UAG琥珀型终止密码子，得到突变体质粒pcDNA3-Smad-39TAG，pcDNA3-Smad-122TAG和 pcDNA3-Smad-133TAG，优选所述突变体序列如SEQ ID NO：4-6所示；
    (3)用突变体质粒转染稳定细胞系HEK293-PYL，加入非天然氨基酸培养1-96小时，优选12-72小时，最优选48小时后提取蛋白，经western blot检测到全长Smad蛋白，根据全长Smad蛋白表达量的多少评价通读效率。
  16. 引物对，其特征在于，所述序列为PCMV-UAG-UAA-for：TGTAGATCGAATGGACTTTAAATCCGTTCAGCCGG和PCMV-UAG-UAA-rev：CCGGCTGAACGGATTTAAAGTCCATTCGATCTACA
    或
    PCMV-UAG-UGA-for：CATGTAGATCGAATGGACTTCAAATCCGTTCAGCCGGGTT和PCMV-UAG-UGA-rev：AACCCGGCTGAACGGATTTGAAGTCCATTCGATCTACATG。
PCT/CN2017/075577 2016-03-10 2017-03-03 利用优化的基因密码子扩展系统通读提前终止密码子疾病中的截短蛋白 Ceased WO2017152808A1 (zh)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111850020A (zh) * 2019-04-25 2020-10-30 苏州鲲鹏生物技术有限公司 利用质粒系统在蛋白中引入非天然氨基酸
CN115261344A (zh) * 2022-08-29 2022-11-01 北京大学 基于非天然氨基酸的离子液体、其制备方法及应用

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW202449155A (zh) * 2018-07-27 2024-12-16 美商薩羅塔治療公司 用於肌肉萎縮症之外顯子跳躍寡聚物
CN110835633B (zh) * 2018-08-13 2021-10-01 北京大学 利用优化的基因密码子扩展系统制备ptc稳定细胞系及应用
CN111849929B (zh) * 2019-04-30 2021-05-11 苏州鲲鹏生物技术有限公司 高效引入赖氨酸衍生物的氨酰基—tRNA合成酶
AU2020295571B2 (en) * 2019-06-21 2026-03-26 Trustees Of Boston College Enhanced platforms for unnatural amino acid incorporation in mammalian cells
CN112553239B (zh) * 2019-09-25 2023-10-13 深圳华大生命科学研究院 基于非天然氨基酸的基因组重排调控系统和方法
CN111304234A (zh) * 2020-02-27 2020-06-19 江南大学 一种适用于枯草芽孢杆菌的非天然氨基酸利用工具
CN113699124B (zh) * 2021-09-08 2022-04-12 北京大学 一种含非天然氨基酸蛋白的制备方法
CN115074330B (zh) * 2022-04-19 2023-12-29 中国医学科学院医学生物学研究所 一种基于遗传密码子扩展技术的改造Vero细胞系
CN114908066B (zh) * 2022-05-17 2024-01-23 杭州嵌化合生医药科技有限公司 一种正交翻译系统及其在再分配密码子恢复ptc疾病中功能蛋白表达方面的应用
CN116804187A (zh) * 2023-07-24 2023-09-26 中国农业科学院兰州兽医研究所 一种复制缺陷型口蹄疫病毒、构建方法及应用
CN118389493B (zh) * 2024-05-10 2025-04-11 北京大学 一种定点修饰的工程化tRNA及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102504022A (zh) * 2011-11-30 2012-06-20 苏州元基生物技术有限公司 含有保护赖氨酸的胰岛素原及使用其制备胰岛素的方法
CN102838663A (zh) * 2011-06-23 2012-12-26 北京大学 定点突变和定点修饰的病毒膜蛋白、其制备方法及其应用
WO2015100277A2 (en) * 2013-12-23 2015-07-02 University Of Rochester Methods and compositions for ribosomal synthesis of macrocyclic peptides

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5214080A (en) * 1987-05-15 1993-05-25 Dainippon Ink And Chemicals, Inc. Process for producing nonaqueous dispersion-type resin
EP1996713B1 (en) * 2006-03-20 2012-08-29 Max-Delbrück-Centrum für Molekulare Medizin (MDC) Use of pol iii promoters for controlled expression of therapeutic proteins

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102838663A (zh) * 2011-06-23 2012-12-26 北京大学 定点突变和定点修饰的病毒膜蛋白、其制备方法及其应用
CN102504022A (zh) * 2011-11-30 2012-06-20 苏州元基生物技术有限公司 含有保护赖氨酸的胰岛素原及使用其制备胰岛素的方法
WO2015100277A2 (en) * 2013-12-23 2015-07-02 University Of Rochester Methods and compositions for ribosomal synthesis of macrocyclic peptides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GUO, NINI ET AL.: "tRNA-directed Transcription Antitermination", PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS, vol. 24, no. 5, 31 December 1997 (1997-12-31), pages 392 - 396, ISSN: 1000-3282 *
ROY, B. ET AL.: "Nonsense suppression by near-cognate tRNAs employs alternative base pairing at codon positions 1 and 3", PNAS, vol. 112, no. 10, 10 March 2015 (2015-03-10), pages 3038 - 3043, XP055415192, ISSN: 0027-8424 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111850020A (zh) * 2019-04-25 2020-10-30 苏州鲲鹏生物技术有限公司 利用质粒系统在蛋白中引入非天然氨基酸
CN115261344A (zh) * 2022-08-29 2022-11-01 北京大学 基于非天然氨基酸的离子液体、其制备方法及应用

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