WO2017152809A1 - 抑制性tRNA通读提前终止密码子疾病中的截短蛋白 - Google Patents
抑制性tRNA通读提前终止密码子疾病中的截短蛋白 Download PDFInfo
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- WO2017152809A1 WO2017152809A1 PCT/CN2017/075581 CN2017075581W WO2017152809A1 WO 2017152809 A1 WO2017152809 A1 WO 2017152809A1 CN 2017075581 W CN2017075581 W CN 2017075581W WO 2017152809 A1 WO2017152809 A1 WO 2017152809A1
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Definitions
- the invention belongs to the field of biopharmaceutics, and particularly relates to the use of a truncated protein for constructing a non-sense mutant of a causative gene by constructing an inhibitory tRNA, thereby producing a full-length functional protein in a mammalian cell, thereby restoring the normal structure and function of the mutant.
- the present invention mainly relates to the construction of an inhibitory tRNA corresponding to three stop codons, which reads dystrophin protein in mammalian cells and reads the non-sense mutant protein in tumor cells, and the effect is remarkable.
- 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.
- Nonsense mutations in the DMD gene are 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.
- Duchenne The incidence of muscular dystrophy in live births is 1/6300 to 1/3500 [Dooley J. et al. 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.
- the 61 codons in the human genome can be recognized by tRNA, encoding 20 amino acids.
- the three stop codons (UAG, UAA, UGA) have no corresponding tRNA recognition, and do not encode amino acids, thus terminating translation.
- studies have found that there is a tRNA that recognizes the stop codon, so that the stop codon encodes an amino acid, and protein translation proceeds normally.
- a tRNA capable of recognizing a stop codon is a nonsense mutation inhibitory tRNA.
- Inhibitory tRNAs are widely available, and inhibitory tRNAs are found in both plant and animal cells. However, since suppressive tRNA is extremely small in cells, inhibitory tRNA is not easily detected.
- the nonsense mutation-inhibiting tRNA is produced by a normal coding amino acid tRNA anti-codon loop base mutation, and the mutated tRNA recognizes the stop codon and is fully complementary to the stop codon, while still carrying the amino acid, capable of prematurely terminating the codon Insert a specific amino acid and read the nonsense mutation. Based on the ability of inhibitory tRNA to read through the meaning of nonsense mutations, it has been reported in the literature to use inhibitory tRNA to read prokaryotic and eukaryotic cells containing proteins with premature stop codons to restore protein expression. Since approximately 30% of human genetic diseases have PTCs, it is still unclear whether the suppressive tRNAs are used for the study of human genetic disease-associated proteins.
- inhibitory tRNAs to extend the truncated proteins of pathogenic gene nonsense mutants, It is important to produce full-length functional proteins in mammalian cells to restore the normal structure and function of the mutant.
- inhibitory tRNA can restore the expression of non-sense mutant proteins, it is still unclear what the difference in the efficiency of different inhibitory tRNA readings. Therefore, it is important to construct a variety of inhibitory tRNAs to compare the efficiency of read-through in mammalian cells.
- the inventors have constructed and studied 19 nonsense mutation-inhibiting tRNAs (sequences such as SEQ ID NOs: 1-19), and the inhibitory tRNA carries the corresponding amino acid and is fully complementary to the premature stop codon.
- Nonsense mutations restore the expression of pathogenic proteins in PTC diseases and compare the most efficient inhibitory tRNAs with nonsense mutations.
- the inventors first identified 19 amino acid codons with higher frequency of nonsense mutations in human hereditary diseases. The corresponding tRNA anticodon loop bases recognizing these 19 codons were changed, and a nonsense mutant suppressor tRNA was constructed by SOE PCR, and a 7sk promoter was ligated at the 5' end of the suppressor tRNA.
- the inhibitory tRNA and the dystrophin protein gene containing the premature stop codon were transfected into 293T cells to restore dystrophin protein expression in the mammalian cells.
- the dual luciferase reporter gene containing the stop codon and the GFP reporter gene were used to compare the different inhibitory tRNAs to improve the reading efficiency.
- the most effective inhibitory tRNAs were Amber inhibitory tRNA (Gln); Ocher inhibitory tRNA (Gln); Opal inhibitory tRNA (Arg).
- the invention relates to a method of rapidly constructing an inhibitory tRNA, wherein the method comprises the steps of:
- the first step PCR uses the corresponding primers to synthesize and amplify the inhibitory tRNA and amplify the 7sk promoter sequence;
- inhibitory tRNA is obtained by tRNA anti-codon loop mutation.
- the 7sk promoter sequence is the sequence shown in Table 1.
- the primer is selected from the sequences shown in Table 2.
- the invention features a method of screening for an inhibitory tRNA, comprising:
- the host cell may be a prokaryotic cell, such as an E. coli cell, an insect cell, or a eukaryotic cell, such as a yeast cell, a mammalian cell, a tumor cell.
- a prokaryotic cell such as an E. coli cell, an insect cell, or a eukaryotic cell, such as a yeast cell, a mammalian cell, a tumor cell.
- the inhibitory tRNA obtained by the method is selected from the group consisting of the inhibitory tRNAs set forth in SEQ ID NOs: 1-19.
- the mutein is selected from the group consisting of a dual luciferase reporter protein, a GFP protein, a dystrophin protein, an STK11 protein, and an EPHB2 protein.
- the invention relates to an inhibitory tRNA obtained by the method of any of the aspects of the invention.
- inhibitory tRNA of any of the aspects of the invention wherein the inhibitory tRNA is selected from the group consisting of the inhibitory tRNAs set forth in SEQ ID NOs: 1-19.
- the invention relates to a plasmid, vector or kit for a tRNA of any of the aspects of the invention.
- kits which comprises an inhibitory tRNA having the sequence set forth in SEQ ID NOs: 1-19.
- a kit according to any aspect of the present invention which comprises Amber inhibitory tRNA (Gln) corresponding to SEQ ID NO: 1; Ocher inhibitory tRNA (Gln) corresponds to SEQ ID NO: 14; Opal inhibitory tRNA (Arg Corresponding to SEQ ID NO: 7.
- the invention relates to the use of a tRNA, plasmid, vector or kit of any of the aspects of the invention for the manufacture of a medicament for the treatment of a genetic disease or cancer, wherein the genetic disease or cancer is due to a genetic nonsense mutation
- 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 , Heller's syndrome, family black idiots, stomach cancer, lung cancer.
- the invention relates to methods for assessing the efficiency of an inhibitory tRNA reading a nonsense mutation. Law, which includes:
- the reporter gene is subjected to point mutation to obtain mutants containing three premature stop codons of UAG, UAA and UGA, respectively, and ligated to an appropriate vector;
- the reporter gene is selected from the group consisting of a dual luciferase reporter protein, a GFP protein, a dystrophin protein, an STK11 protein, and an EPHB2 protein.
- the invention relates to a method for restoring a disease-causing gene of a monogenic genetic disease and a truncated protein expression of a tumor suppressor gene non-sense mutant in a tumor cell, comprising a tRNA or plasmid or vector of any aspect of the invention Introduction into a cell or organism containing a non-sense mutant protein, preferably using a kit of any of the aspects of the invention.
- the invention relates to the use of an inhibitory tRNA to read a single gene disease and a nonsense mutation site in a tumor cell to obtain a full length functional protein.
- the inhibitory tRNA according to any of the aspects of the present invention, which comprises an inhibitory tRNA species corresponding to an amino acid in which a nonsense mutation may occur in a hereditary disease, that is, all inhibitory tRNAs corresponding to 20 amino acids. It is characterized in that the inhibitory tRNA is completely complementary to the stop codon and the inhibitory tRNA is obtained by tRNA anti-codon loop mutation.
- An inhibitory tRNA according to any aspect of the invention is characterized in that the 5' end of the inhibitory tRNA is linked to a 7sk promoter.
- the first step PCR uses the corresponding primers to synthesize and amplify the inhibitory tRNA and amplify the 7sk promoter sequence.
- the inhibitory tRNA and the 7sk promoter were ligated by PCR, and the ligation product was amplified, and finally 19 inhibitory tRNAs carrying the 7sk promoter were obtained.
- the inhibitory tRNA obtained by the method of any aspect of the invention corresponds to SEQ ID NO: 1; stRNATyr-UAG, sequence pair SEQ ID NO: 2; stRNALys-UAG sequence corresponds to SEQ ID NO: 3; stRNALeu-UAG, sequence corresponds to SEQ ID NO: 4; stRNAGlu-UAG, sequence corresponds to SEQ ID NO: 5; stRNATrp-UAG, sequence corresponds to SEQ ID NO:6; stRNAArg-UGA, the sequence corresponds to SEQ ID NO:7; stRNAGln-UGA, the sequence corresponds to SEQ ID NO:8; stRNATrp-UGA, the sequence corresponds to SEQ ID NO:9; stRNAGly-UGA, the sequence corresponds to SEQ ID NO: 10; stRNACys-UGA, the sequence corresponds to SEQ ID NO: 10; stRNACys-UGA, the
- An inhibitory tRNA of any aspect of the invention is ligated to a Bjmu vector as set forth in SEQ ID NO: 20.
- An inhibitory tRNA of any of the aspects of the invention which is Bjmu-stRNAGln-UAG; Bjmu-stRNATyr-UAG; Bjmu-stRNALys-UAG; Bjmu-stRNALeu-UAG; Bjmu-stRNAGlu-UAG; Bjmu-stRNATrp-UAG; Bjmu-stRNAArg-UGA; Bjmu-stRNATrp-UGA; Bjmu-stRNAGly-UGA; Bjmu-stRNACys-UGA; Bjmu-stRNALeu-UGA; Bjmu-stRNASer-UGA; Bjmu-stRNAGln-UAA; Bjmu- stRNATyr-UAA; Bjmu-stRNALys-UAA; Bjmu-stRNAGlu-UAA; Bjmu-st
- inhibitory tRNA of any aspect of the invention which differs in the efficiency of reading nonsense mutations, is obtained by the following steps:
- the original sequence is mutated by GFP fluorescent gene pcDNA3.1-GFP as shown in SEQ ID NO: 21 to obtain three premature stop codons of UAG, UAA and UGA, respectively, to obtain pcDNA3.1-GFP-39TAG; pcDNA3.1-GFP-39TAA; pcDNA3.1-GFP-39TGA vector.
- the method for inhibiting the expression of a truncated protein of a disease-inducing gene of a monogenic genetic disease and a non-sense mutant of a tumor suppressor gene in a tumor cell comprising the steps of:
- the Dp71b gene having the sequence of SEQ ID NO: 23 is mapped to a position requiring mutation according to the position of the nonsense mutation in the human DMD disease, and the DMD gene sequence in the human DMD disease is mimicked.
- inhibitory tRNA can restore pathogenic genes of monogenic genetic diseases and tumor suppressor gene nonsense mutants in tumor cells The truncated protein is expressed and the efficiency of different inhibitory tRNA recovery is different.
- an inhibitory tRNA is constructed and DMD disease-associated protein dystrophin protein expression is restored in 293T cells, mainly by: (1) constructing an expression vector of 19 inhibitory tRNAs containing a 7sk promoter Bjmu-stRNAGln-UAG; Bjmu-stRNALyr-UAG; Bjmu-stRNALeu-UAG; Bjmu-stRNALlu-UAG; Bjmu-stRNATrp-UAG; Bjmu-stRNAArg-UGA; Bjmu-stRNAGln-UGA; Bjmu- stRNATrp-UGA; Bjmu-stRNAGly-UGA; Bjmu-stRNACys-UGA; Bjmu-stRNALeu-UGA; Bjmu-stRNASer-UGA; Bjmu-stRNAGln-UAA
- the principle of inhibitory tRNA read-through nonsense mutations is: (1) In the normal translation process of cells, the early stop codon is recognized by the first type of peptide chain release factor eRF1, while the normal tRNA cannot recognize the stop codon, and eRF3 is a class. GTPase, which relies on ribosomes and the first class of peptide chain release factors, cooperates with eRF1 to promote release of the peptide chain from the ribosome, and the translation process is terminated (Zhouravleva, G. et al. EMBO J, 1995, 14, 4065-72.).
- the constructed suppressor tRNA is a normal tRNA transformed by an anti-codon loop, and its anti-codon loop can
- the stop codons UAG, UAA, and UGA are fully complementary paired, and the eRF1 competes to recognize the premature stop codon, and the tRNA that changes the anti-codon loop can still carry the corresponding amino acid. Therefore, the inhibitory tRNA inserts the amino acid at the premature stop codon position.
- the translation process continues, reading through the nonsense mutations; (2) the constructed inhibitory tRNA 5' segment is ligated with the 7sk promoter, which can initiate the expression of inhibitory tRNA in mammalian cells and ultimately restore protein expression.
- the rapid construction of any of the inhibitory tRNAs with the 7sk promoter is achieved using the SOE PCR method.
- the principle is that the inhibitory tRNA is only a single base substitution of normal tRNA, the size is generally about 80bp, and the length is small.
- PCR was used to synthesize inhibitory tRNA and 7sk promoter sequences respectively.
- PCR was used to link the inhibitory tRNA to the 7sk promoter to synthesize the inhibitory tRNA with the 7sk promoter.
- 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; pcDNA3.
- the 1-GFP-39TGA vector was used to determine the efficiency of GFP fluorescence in 293T cells, and the efficiency of inhibitory tRNA read-through was determined.
- the most effective inhibitory tRNAs for UAG, UAA and UGA were identified as Amber inhibitory tRNA (Gln); Ocher inhibitory tRNA (Gln); Opal inhibitory tRNA (Arg).
- the inhibitory tRNA 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 normal Dp71b sequence to mimic DMD gene sequence in human DMD disease, Dp71b 3115TAG containing premature stop codon UAG, mutation to c.9346C>T, including premature termination UAA codon of Dp71b 3216TAA, mutation c.9651C>a; containing a UGA premature termination codon Dp71b 3112TGA, mutated c.9337C> T.
- the mutated Dp71b protein granules were co-transfected into different 293T cells with different inhibitory tRNAs to restore the expression of Dp71b.
- the inhibitory tRNA is used to read a nonsense mutation site of a tumor suppressor gene in a tumor cell.
- Bjmu-stRNAGln-UAG was transfected into tumor cell lines A549 and DU145 (sense mutation of STK11 in human lung cancer cell A 549 genome c.109C>T, p.Q37X, as stop codon UAG; human prostate cancer cell DU 145 genome
- 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
- the efficiency of inhibitory tRNA read-through can be reflected by measuring the fluorescence intensity of this gene relative to Renila fluorescence intensity.
- Dp71b protein particle Dp71b 3115TAG ; Dp71b 3216TAA ; Dp71b 3112TGA containing premature stop codon UAG, UAA, UGA.
- the unmutated Dp71b sequence is set forth in SEQ ID NO:23.
- FIG. 1 Construction of an inhibitory tRNA plasmid
- Inhibitory tRNA was digested with BamHI and Bgl II, Bjmu vector was digested with BamHI, and the digested product was ligated to obtain a Bjmu vector to which 7sk-inhibitory tRNA was ligated.
- double luciferase is linked with a linker containing a stop codon, and the wild-type GFP gene mutates 39 amino acid to a premature stop codon by point mutation;
- Figure 3 Inhibitory tRNA restores PTC disease and expression of nonsense mutant proteins in tumor cells
- Inhibitory tRNA can restore Dp71b protein expression, but the recovery efficiency is different;
- inhibitory tRNA is, Amber Inhibitory tRNA: inhibitory tRNA (Gln/UAG), inhibitory tRNA (Tyr/UAG), inhibitory tRNA (Lys/UAG), inhibitory tRNA (Leu/UAG), inhibitory tRNA (Glu/UAG), inhibition tRNA (Trp/UAG); Opal inhibitory tRNA: inhibitory tRNA (Arg/UGA), inhibitory tRNA (Gln/UGA), inhibitory tRNA (Trp/UGA), inhibitory tRNA (Gly/UGA), inhibition tRNA (Cys/UGA), inhibitory tRNA (Leu/UGA), inhibitory tRNA (Ser/UGA); Ocher inhibitory tRNA
- the inhibitory tRNA (Lys/UAG) was synthesized by sequence synthesis, and the remaining 13 were synthesized by SOE PCR and the 7sk promoter was ligated at the 5' end, and the three were obtained by the obtained suppressor tRNA point mutation.
- Point mutation primer name Primer sequence (5'-3' direction) 7sk-Gln-UGA for Ctcggatcgctggatttgaagtccagagtgctaac 7sk-Gln-UGA rev Gttagcactctggacttcaaatccagcgatccgag 7sk-Tyr-UAA for Gcgacctaaggatctaaagtcctccgctctacc 7sk-Tyr-UAA rev Ggtagagcggaggactttagatccttaggtcgc 7sk-Trp-UAG-for Gcaacggcagcgcgtctgactctagatcagaaggt UAG-Trp-rev Accttctgatctagagtcagacgcgctgccgttgcgttgccgttgccgcgttgccgcgt
- Example 2 Detection of 19 inhibitory tRNA read-through efficiencies using a dual fluorescein reporter gene and a point-mutated GFP reporter gene
- 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:21.
- 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 inhibitory tRNA vector was mixed with the dual fluorescein reporter gene pGL4-2luc-TAG; pGL4-2luc-TAA; pGL4-2luc-TGA plasmid in a ratio of 1:2 in groups of 5, and then transfected with megatrans 1.0 Mix 1:3, add 293T cells together, change the solution after 6 hours, continue to culture for 48 hours in 37 ° C, 5% CO 2 incubator, lyse the cells, add luciferase substrate to the cell lysate, test Fluorescence reading. The result is shown in Figure 2b. After the addition of the inhibitory tRNA, a full-length active mutant firefly luciferase protein can be obtained.
- the most effective inhibitory tRNAs for UAG, UAA and UGA were identified as Amber inhibitory tRNA (Gln); Ocher inhibitory tRNA (Gln); Opal inhibitory tRNA (Arg).
- Bjmu-stRNAGln-UAG and pGL4-2luc-TAG 2 Bjmu-stRNATyr-UAG and pGL4-2luc-TAG 3
- Bjmu-stRNALys-UAG and pGL4-2luc-TAG 4 Bjmu-stRNALeu-UAG and pGL4-2luc-TAG 5
- Bjmu-stRNAGlu-UAG and pGL4-2luc-TAG 6 Bjmu-stRNATrp-UAG and pGL4-2luc-TAG 7
- Bjmu-stRNAArg-UGA and pGL4-2luc-TGA 8 Bjmu-stRNAGln-UGA and pGL4-2luc-TGA 9
- Bjmu-stRNATrp-UGA and pGL4-2luc-TGA 10 Bjmu-stRNAGly-UGA and pGL4-2luc-TGA 11 Bj
- the pcDNA3.1-GFP-39TXX obtained in the first step of Example 2, and the 19 inhibitory tRNA plasmids of the step 3 of Example 1 were transfected according to the grouping of Table 6 according to the transfection method described in Step 2 of Example 2 to 293T cells were observed for fluorescence fluorescence microscopy after 48 hours, and the results are shown in Figure 2c.
- the most effective inhibitory tRNAs for UAG, UAA and UGA were Amber inhibitory tRNA (Gln); Ocher inhibitory tRNA (Gln); Opal inhibitory tRNA (Arg).
- the Dp71b sequence of the Dystrophin protein is shown in SEQ ID NO: 23, and the inventors performed a point mutation 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.
- the Dp71b 3115TAG , Dp71b 3216TAA , Dp71b 3112TGA plasmid obtained in the first step of Example 3 and the corresponding inhibitory tRNA were transfected into 293T cells according to the method described in Step 2 of Example 2, and the protein was extracted after 48 hours of culture, western.
- the detection of the full-length dystrophin protein by blot detection (anti-dystrophin, anti-dystrophin C-terminal antibody, Cat. No. 12715-1-AP) was shown in Figure 3a. It was demonstrated that the inhibitory tRNA can read through different types of premature stop codons and restore the expression of disease proteins.
- Example 4 Inhibitory tRNA read-through early stop codons on the genome of tumor cell lines
- 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 Bjmu-stRNAGln-UAG plasmid was mixed with the transfection reagent megatrans1.0 at a ratio of 1:3, and transfected into A 549 and DU 145 cells respectively. After 6 hours, the cells were changed, and the cells were placed in an incubator at 37 ° C, 5% CO 2 . Proteins were extracted after 48 hours of incubation, and Western blot analysis (primary antibodies anti-STK11 and anti-EPHB2, respectively) to STK11 and EPHB2 production of full-length proteins, as shown in Figures 3b and 3c. Verify that inhibition is able to read through the endogenous genome The codon was stopped early to restore the expression of the tumor suppressor gene protein.
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Abstract
提供了构建抑制性tRNA的方法以及对应三种终止密码子的19种抑制性tRNA，含有上述tRNA的质粒、载体或试剂盒。还提供了上述tRNA、质粒、载体或试剂盒在制备用于治疗由于基因无义突变引起的遗传病或癌症的药物中的用途，评价抑制性tRNA通读无义突变的效率的方法以及恢复单基因遗传病的致病基因和肿瘤细胞内的肿瘤抑制基因无义突变体的截断蛋白的表达方法。
Description
本发明属于生物制药领域，具体涉及利用构建抑制性tRNA延长致病基因无义突变体的截短蛋白，使哺乳动物细胞中产生全长有功能的蛋白，从而恢复突变体的正常结构和功能。本发明主要涉及构筑对应三种终止密码子的抑制性tRNA，在哺乳动物细胞中通读dystrophin蛋白并在肿瘤细胞通读无义突变蛋白，作用显著。
无义突变及其引起的遗传性疾病
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，从而引发相应的遗传性疾病。据统计，大约有11.2％的单基因遗传性疾病会产生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.et al.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.et al.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。
抑制性tRNA通读无义突变
人类基因组中的61种密码子可以被tRNA识别，编码20种氨基酸，三种终止密码子(UAG、UAA、UGA)没有相应的tRNA识别，不编码氨基酸，进而终止翻译。但是有研究发现，存在可以识别终止密码子的tRNA，使终止密码子编码氨基酸，蛋白翻译正常进行。这种能够识别终止密码子的tRNA，即为无义突变抑制性tRNA。抑制性tRNA存在广泛，无论在植物细胞还是动物细胞中，都发现了抑制性tRNA。但由于抑制性tRNA在细胞中含量极少，因此抑制性tRNA不易被检测到。
无义突变抑制性tRNA由正常编码氨基酸tRNA反密码子环碱基突变产生，突变后的tRNA能识别终止密码子并与终止密码子完全互补配对，同时仍能携带氨基酸，能够在提前终止密码子处插入特定氨基酸，通读无义突变。基于抑制性tRNA能够通读无义突变的特性，有文献报道应用抑制性tRNA通读原核和真核细胞中含有提前终止密码子的蛋白，恢复蛋白表达。由于大约30％人类遗传性疾病中存在PTC，但抑制性tRNA对于人类遗传性疾病相关蛋白通读情况研究仍不清楚，因此，利用构建抑制性tRNA延长致病基因无义突变体的截短蛋白，使哺乳动物细胞中产生全长有功能的蛋白，从而恢复突变体的正常结构和功能显得十分重要。另一方面，虽然抑制性tRNA能够恢复含无义突变蛋白表达，但是，不同抑制性tRNA通读效率有何差异，仍不得而知。因此，构建多种抑制性tRNA，比较在哺乳动物细胞中通读效率差异，需找高效抑制性tRNA也十分重要。
发明内容
发明人经过对现有技术的思考和研究，构建19种无义突变抑制性tRNA(序列如SEQ ID NO：1-19)，抑制性tRNA携带对应氨基酸并与提前终止密码子完全互补配对，通读无义突变，恢复PTC疾病中致病蛋白表达，并比较得通读无义突变效率最高的抑制性tRNA。发明人首先确定了人类遗传性疾病中19种无义突变频率较高的氨基酸密码子，
改变识别这19种密码子的相应tRNA反密码子环碱基，通过SOE PCR的方法构建无义突变抑制性tRNA，并在抑制性tRNA 5’端连接7sk启动子。再将抑制性tRNA与含有提前终止密码子的dystrophin蛋白基因转染至293T细胞，在哺乳细胞中恢复dystrophin蛋白表达。同时，应用含有终止密码子的双荧光素酶报告基因和GFP报告基因比较不同抑制性tRNA促进通读效率，比较得到通读效率最高的抑制性tRNA分别为Amber抑制性tRNA(Gln)；Ocher抑制性tRNA(Gln)；Opal抑制性tRNA(Arg)。
本发明的优点可体现在如下中的一个或几个：
1、通过SOE PCR的方法，实现任意一种抑制性tRNA的快速构建。
2、获得了三种能够高效通读无义突变的抑制性tRNA——Amber抑制性tRNA(Gln)；Ocher抑制性tRNA(Gln)；Opal抑制性tRNA(Arg)。
3、利用多种抑制性tRNA，实现通读单基因遗传性疾病和肿瘤中的无义突变，恢复截短蛋白的正常结构和功能。
在一方面，本发明涉及快速构建抑制性tRNA的方法，其中所述方法包括下列步骤：
(1)设计覆盖全部抑制性tRNA并部分互补的上下游引物，同时设计7sk基因PCR引物，7sk基因PCR下游引物能同时与7sk和抑制性tRNA互补；
(2)第一步PCR分别使用对应的引物合成并扩增抑制性tRNA及扩增7sk启动子序列；
(3)第二步PCR连接抑制性tRNA与7sk启动子，并扩增连接产物，最终得到带有7sk启动子的抑制性tRNA；
其中所述抑制性tRNA由tRNA反密码子环突变得到。
根据本发明的任一方面，其中所述7sk启动子序列为表1所示的序列。
根据本发明的任一方面，其中所述引物选自表2所示的序列。
在一方面，本发明涉及筛选抑制性tRNA的方法，其包括：
(1)确定人类遗传性疾病中19种无义突变频率较高的氨基酸密码子，改变识别这19种密码子的相应tRNA反密码子环碱基；
(2)构建无义突变抑制性tRNA，并在抑制性tRNA 5’端连接7sk启动子；
(3)将抑制性tRNA与含有提前终止密码子的突变蛋白基因转染至宿主动物细胞，在宿主细胞中恢复突变蛋白正常表达；
(4)比较不同抑制性tRNA促进通读效率，比较得到通读效率高的抑制性tRNA。
根据本发明的任一方面，其中，所述宿主细胞可以是原核细胞，诸如大肠杆菌细胞、昆虫细胞，或者真核细胞，诸如酵母细胞、哺乳动物细胞、肿瘤细胞。
根据本发明的任一方面，其中，所述方法得到的抑制性tRNA选自SEQ ID NO：1-19所示的抑制性tRNA。
根据本发明的任一方面，其中，所述突变蛋白选自双荧光素酶报告基因蛋白、GFP蛋白、dystrophin蛋白、STK11蛋白和EPHB2蛋白。
在一方面，本发明涉及本发明的任一方面的方法得到的抑制性tRNA。
本发明的任一方面的抑制性tRNA，其中，所述抑制性tRNA选自序列如SEQ ID NO：1-19所示的抑制性tRNA。
在一方面，本发明涉及本发明的任一方面的tRNA的质粒、载体或试剂盒。
本发明的任一方面的试剂盒，其中，其包含序列如SEQ ID NO：1-19所示的抑制性tRNA。
本发明的任一方面的试剂盒，其特征在于，其包含Amber抑制性tRNA(Gln)对应SEQ ID NO：1；Ocher抑制性tRNA(Gln)对应SEQ ID NO：14；Opal抑制性tRNA(Arg)对应SEQ ID NO：7。
在一方面，本发明涉及本发明的任一方面的tRNA、质粒、载体或试剂盒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。
本发明的任一方面的用途，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，本发明涉及评价抑制性tRNA通读无义突变的效率的方
法，其包括：
(1)将报告基因进行点突变，得到分别含有UAG、UAA、UGA三种提前终止密码子的突变体，并连接适当的载体；
(2)将步骤(1)获得的包含突变报告基因的载体与不同抑制性tRNA分别共转染至宿主细胞之中；
(3)检测报告基因，根据报告基因检测结果确定抑制性tRNA通读效率。
本发明的任一方面的方法，其中所述报告基因选自双荧光素酶报告基因蛋白、GFP蛋白、dystrophin蛋白、STK11蛋白和EPHB2蛋白。
在一方面，本发明涉及恢复单基因遗传病的致病基因和肿瘤细胞内的肿瘤抑制基因无义突变体的截短蛋白表达的方法，包括将本发明的任一方面的tRNA或质粒、载体导入含有无义突变体蛋白的细胞或生物体内，优选地，使用本发明的任一方面的试剂盒进行。
在一方面，本发明涉及利用抑制性tRNA通读单基因病和肿瘤细胞中无义突变位点，获得全长有功能的蛋白。
本发明的任一方面的所述的抑制性tRNA，其包含遗传性疾病中可能发生无义突变的氨基酸对应的抑制性tRNA种类，即对应20种氨基酸的所有抑制性tRNAs。其特征在于，抑制性tRNA与终止密码子完全互补配对，抑制性tRNA由tRNA反密码子环突变得到。
本发明的任一方面的抑制性tRNA，其特征在于所述的抑制性tRNA5’端连接7sk启动子。
本发明的任一方面的抑制性tRNA，其是通过下述SOE PCR的方法获得的：
(1)设计覆盖全部抑制性tRNA并部分互补的上下游引物，同时设计7sk基因PCR引物，7sk基因PCR下游引物能同时与7sk和抑制性tRNA互补。
(2)第一步PCR分别使用对应的引物合成并扩增抑制性tRNA及扩增7sk启动子序列。
(3)第二步PCR连接抑制性tRNA与7sk启动子，并扩增连接产物，最终得到19种带有7sk启动子的抑制性tRNA。
本发明的任一方面的方法所获得所述抑制性tRNA，其分别stRNAGln-UAG，序列对应SEQ ID NO：1；stRNATyr-UAG，序列对
应SEQ ID NO：2；stRNALys-UAG序列对应SEQ ID NO：3；stRNALeu-UAG，序列对应SEQ ID NO：4；stRNAGlu-UAG，序列对应SEQ ID NO：5；stRNATrp-UAG，序列对应SEQ ID NO：6；stRNAArg-UGA，序列对应SEQ ID NO：7；stRNAGln-UGA，序列对应SEQ ID NO：8；stRNATrp-UGA，序列对应SEQ ID NO：9；stRNAGly-UGA，序列对应SEQ ID NO：10；stRNACys-UGA，序列对应SEQ ID NO：11；stRNALeu-UGA，序列对应SEQ ID NO：12；stRNASer-UGA，序列对应SEQ ID NO：13；stRNAGln-UAA，序列对应SEQ ID NO：14；stRNATyr-UAA，序列对应SEQ ID NO：15；stRNALys-UAA，序列对应SEQ ID NO：16；stRNAGlu-UAA，序列对应SEQ ID NO：17；stRNALeu-UAA，序列对应SEQ ID NO：18；stRNASer-UAA，序列对应SEQ ID NO：19。
本发明的任一方面的抑制性tRNA，酶切连接至序列如SEQ ID NO：20所示的Bjmu载体上。
本发明的任一方面的抑制性tRNA，其分别是Bjmu-stRNAGln-UAG；Bjmu-stRNATyr-UAG；Bjmu-stRNALys-UAG；Bjmu-stRNALeu-UAG；Bjmu-stRNAGlu-UAG；Bjmu-stRNATrp-UAG；Bjmu-stRNAArg-UGA；Bjmu-stRNAGln-UGA；Bjmu-stRNATrp-UGA；Bjmu-stRNAGly-UGA；Bjmu-stRNACys-UGA；Bjmu-stRNALeu-UGA；Bjmu-stRNASer-UGA；Bjmu-stRNAGln-UAA；Bjmu-stRNATyr-UAA；Bjmu-stRNALys-UAA；Bjmu-stRNAGlu-UAA；Bjmu-stRNALeu-UAA；Bjmu-stRNASer-UAA。
本发明的任一方面的抑制性tRNA，其通读无义突变的效率差异，是通过下述步骤获得的：
(1)将原序列如SEQ ID NO：21所示的GFP荧光基因pcDNA3.1-GFP进行点突变，得到分别含有UAG、UAA、UGA三种提前终止密码子得到pcDNA3.1-GFP-39TAG；pcDNA3.1-GFP-39TAA；pcDNA3.1-GFP-39TGA载体。
(2)将序列如SEQ ID NO：22所示的荧光素酶报告基因pGL4-2luc-TAG；pGL4-2luc-TAA；pGL4-2luc-TGA，与不同抑制性tRNA分别共转染至293T细胞之中。另一方面，将pcDNA3.1-GFP-39TAG；pcDNA3.1-GFP-39TAA；pcDNA3.1-GFP-39TGA载体也同不同抑制性
tRNA分别共转染至293T细胞之中。
(3)检测荧光素酶报告基因Firefly和Renila的荧光读值，根据Firefly相对于Renila的荧光相对值，反应通读效率差异。并根据GFP荧光强度，确定抑制性tRNA通读效率，最终确定通读UAG、UAA、UGA效率最高的抑制性tRNA分别为Amber抑制性tRNA(Gln)；Ocher抑制性tRNA(Gln)；Opal抑制性tRNA(Arg)。
本发明的任一方面的抑制性tRNA，恢复单基因遗传病的致病基因和肿瘤细胞内的肿瘤抑制基因无义突变体的截短蛋白表达的方法，包括步骤：
(1)根据人类DMD疾病中无义突变位置，将序列如SEQ ID NO：23的Dp71b基因对应需要突变的位置，模拟人类DMD疾病中DMD基因序列。
(2)利用点突变技术得到含有提前终止密码子UAG的Dp71b3115TAG，含有提前终止密码子UAA的Dp71b3216TAA；含有提前终止密码子UGA的Dp71b3112TGA。
(3)将不同的抑制性tRNA与突变后的Dp71b质粒共转染至293T或将抑制性tRNA转染至肿瘤细胞A549和DU145中，适当时间后收集细胞。
(4)检测293T细胞中Dp71b表达情况，A549和DU145中STK11蛋白和全长EPHB2蛋白表达情况，抑制性tRNA能够恢复单基因遗传病的致病基因和肿瘤细胞内的肿瘤抑制基因无义突变体的截短蛋白表达，并且不同抑制性tRNA恢复效率不同。
具体地，在本发明的一个实施方案中，构建抑制性tRNA并在293T细胞中恢复DMD疾病相关蛋白dystrophin蛋白表达，主要通过：(1)构建含有7sk启动子的19种抑制性tRNA的表达载体Bjmu-stRNAGln-UAG；Bjmu-stRNATyr-UAG；Bjmu-stRNALys-UAG；Bjmu-stRNALeu-UAG；Bjmu-stRNAGlu-UAG；Bjmu-stRNATrp-UAG；Bjmu-stRNAArg-UGA；Bjmu-stRNAGln-UGA；Bjmu-stRNATrp-UGA；Bjmu-stRNAGly-UGA；Bjmu-stRNACys-UGA；Bjmu-stRNALeu-UGA；Bjmu-stRNASer-UGA；Bjmu-stRNAGln-UAA；Bjmu-stRNATyr-UAA；
Bjmu-stRNALys-UAA；Bjmu-stRNAGlu-UAA；Bjmu-stRNALeu-UAA；Bjmu-stRNASer-UAA，19种抑制性tRNA对应19种较易发生无义突变的119种氨基酸密码子；(2)构建中间含有终止密码子的双荧光素酶报告系统pGL4-2luc-TAG；pGL4-2luc-TAA；pGL4-2luc-TGA和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病人中无义突变位点，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，构建分别含有提前终止密码子UAG，UAA，UGA的Dp71b蛋白质粒Dp71b3115TAG；Dp71b3216TAA；Dp71b3112TGA；(4)将步骤(1)和步骤(2)中载体分别对应共转染至293T细胞中，利用双荧光素酶和GFP报告系统比较不同抑制性tRNA通读效率差异；(5)将步骤(1)的抑制性tRNA分别与步骤(3)中的含有提前终止密码子的Dp71b蛋白共同转染至293T细胞，western blot方法检测Dp71b表达恢复情况，最终确定多种抑制性tRNA能够恢复无义突变的Dp71b蛋白表达，其中抑制性tRNA(Gln)对于UAG、UAA、UGA三种终止密码子的无义突变均有较高的通读效率，通过双荧光素酶报告系统精确定量抑制性tRNA的通读效率，我们发现stRNAGln-UAG对双荧光素酶报告系统的提前终止密码子UAG最高通读效率为44.7％±1.36％，stRNAGln-UAA对双荧光素酶报告系统的提前终止密码子UAA最高通读效率为30.95％±1.358％，stRNAArg-UGA对双荧光素酶报告系统的提前终止密码子UGA最高通读效率为22.55％±1.39％，远高于文献中报道的通读效率(Ramesh Koukuntla.et a1.J Gene Med， 2013，15，93-101.)；(6)将步骤(1)的抑制性tRNA转染至肿瘤细胞系A549和DU145，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。
抑制性tRNA通读无义突变的原理在于：(1)在细胞正常的翻译过程中，提前终止密码子被第一类肽链释放因子eRF1识别，而正常tRNA不能识别终止密码子，eRF3是一类依赖于核糖体和第一类肽链释放因子的GTPase，协同eRFl促进肽链从核糖体上释放，翻译过程终止(Zhouravleva，G.et al.EMBO J，1995，14，4065-72.)。而构建的抑制性tRNA为正常tRNA通过反密码子环改造而来，其反密码子环能够与
终止密码子UAG、UAA、UGA完全互补配对，与eRF1竞争识别提前终止密码子，改变反密码子环的tRNA仍能携带对应的氨基酸，因此，抑制性tRNA在提前终止密码子位置插入氨基酸，使翻译过程继续进行，通读无义突变；(2)所构建的抑制性tRNA 5’段连有7sk启动子，能够启动抑制性tRNA在哺乳动物细胞中大量表达，最终恢复蛋白表达。
在本发明的一个具体实施方案中，利用SOE PCR方法实现任意一种带有7sk启动子的抑制性tRNA的快速构建。原理在于，抑制性tRNA只是正常tRNA单个碱基的替换，大小一般在80bp左右，长度较小，可以设计部分互补配对的上下游两段引物直接PCR合成，合成的抑制性tRNA进行第二步PCR，连接于7sk启动子3’端。具体地为，设计覆盖全部抑制性tRNA并部分互补的上下游引物，其余引物设计按照SOE PCR常规方法设计。第一步PCR分别合成抑制性tRNA及7sk启动子序列，第二步PCR实现抑制性tRNA与7sk启动子的连接，实现带有7sk启动子的抑制性tRNA的合成。
在本发明的一个具体的实施方案中，利用中间分别含有终止密码子UAG、UAA、UGA的Renila荧光素酶和Firefly酶双荧光素酶报告基因pGL4-2luc-TAG；pGL4-2luc-TAA；pGL4-2luc-TGA，检测不同抑制性tRNA的通读效率。即在293T细胞中转染抑制性tRNA和相应双荧光素酶报告基因，通过分别测定Firefly和Renila的荧光读值，根据Firefly相对于Renila的荧光相对值，反应通读效率差异。同时，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；pcDNA3.1-GFP-39TGA载体，通过测定293T细胞中GFP荧光强度，确定抑制性tRNA通读效率，最终确定通读UAG、UAA、UGA效率最高的抑制性tRNA分别为Amber抑制性tRNA(Gln)；Ocher抑制性tRNA(Gln)；Opal抑制性tRNA(Arg)。
在本发明的一个具体的实施方案中，将抑制性tRNA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，在正常Dp71b序列对应位置进行点突变，模拟人类DMD疾病中DMD基因序列，含有提前终止密码子UAG的Dp71b3115TAG，突变为c.9346C＞T，含有提前终止密码子UAA的Dp71b3216TAA，突变为c.9651C＞A；含有提前终止密码子UGA的Dp71b3112TGA，突变为
c.9337C＞T。将突变后的Dp71b蛋白质粒与不同的抑制性tRNA共转染至293T细胞中，恢复Dp71b的表达。
在本发明的一个具体的实施方案中，将抑制性tRNA用于通读肿瘤细胞中抑癌基因的无义突变位点。将Bjmu-stRNAGln-UAG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.含有7sk启动子的19种抑制性tRNA的表达载体Bjmu-stRNAGln-UAG；Bjmu-stRNATyr-UAG；Bjmu-stRNALys-UAG；Bjmu-stRNALeu-UAG；Bjmu-stRNAGlu-UAG；Bjmu-stRNATrp-UAG；Bjmu-stRNAArg-UGA；Bjmu-stRNAGln-UGA；Bjmu-stRNATrp-UGA；Bjmu-stRNAGly-UGA；Bjmu-stRNACys-UGA；Bjmu-stRNALeu-UGA；Bjmu-stRNASer-UGA；Bjmu-stRNAGln-UAA；Bjmu-stRNATyr-UAA；Bjmu-stRNALys-UAA；Bjmu-stRNAGlu-UAA；Bjmu-stRNALeu-UAA；Bjmu-stRNASer-UAA，其可实现不同抑制性tRNA的过表达。
2.含有终止密码子的双荧光素酶报告基因pGL4-2luc-TAG；pGL4-2luc-TAA；pGL4-2luc-TGA，其序列如SEQ ID NO：22所示。可通过测定该基因的Firely相对于Renila荧光强度，反映抑制性tRNA通读效率。
3.携带Tyr39位分别突变为UAG、UAA、UGA的绿色荧光蛋白报告基因载体pcDNA3.1-GFP-39TAG；pcDNA3.1-GFP-39TAA；pcDNA3.1-GFP-39TGA，该载体可通过荧光强度反映通读效率，未经突变的序列如SEQ ID NO：21所示。
4.含有提前终止密码子UAG，UAA，UGA的Dp71b蛋白质粒Dp71b3115TAG；Dp71b3216TAA；Dp71b3112TGA。未经突变的Dp71b序列如SEQ ID NO：23所示。
图1：抑制性tRNA质粒构建
a：正常tRNA改造反密码子环合成抑制性tRNA；
b：抑制性tRNA通读PTC示意图，携带氨基酸的抑制性tRNA反密码子环与提前终止密码子完全互补配对，通读PTC；
c：SOE PCR方法将7sk启动子连接至抑制性tRNA 5’端；
d：用BamHI和Bgl II双酶切抑制性tRNA，BamHI单酶切Bjmu载体，将酶切产物连接，得到连有7sk-抑制性tRNA的Bjmu载体。
图2：双荧光素酶报告基因及GFP报告基因检测抑制性tRNA通读效率
a：双荧光素酶报告基因及GFP报告基因构建，双荧光素酶中间连有含有终止密码子的linker，野生型GFP基因通过点突变将39为氨基酸突变为提前终止密码子；
b：双荧光素酶报告基因检测不同抑制性tRNA通读效率，不同抑制性tRNA通读效率不同，通读UAG、UAA、UGA效率最高的抑制性tRNA分别为Amber抑制性tRNA(Gln)；Ocher抑制性tRNA(Gln)；Opal抑制性tRNA(Arg)；
c：GFP报告基因荧光强度检测不同抑制性tRNA通读效率，进一步验证了抑制性tRNA不同通读效率。
图3：抑制性tRNA恢复PTC疾病中及肿瘤细胞无义突变蛋白表达
a：抑制性tRNA能够恢复Dp71b蛋白表达，但恢复效率不同；
b：转染抑制性tRNAGln-UAG，A549中STK11蛋白表达恢复；
c：转染抑制性tRNAGln-UAG，DU145中EPHB2蛋白表达恢复。
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1：19种抑制性tRNA的获得
(1)确定19种抑制性tRNA
根据人类PTC疾病突变特点，确定19种可发生无义突变的氨基酸密码子所对应tRNA的序列，改变tRNA反密码子环得到与提前中终止密码子完全互补配对的抑制性tRNA，19种抑制性tRNA分别为，Amber
抑制性tRNA：抑制性tRNA(Gln/UAG)、抑制性tRNA(Tyr/UAG)、抑制性tRNA(Lys/UAG)、抑制性tRNA(Leu/UAG)、抑制性tRNA(Glu/UAG)、抑制性tRNA(Trp/UAG)；Opal抑制性tRNA：抑制性tRNA(Arg/UGA)、抑制性tRNA(Gln/UGA)、抑制性tRNA(Trp/UGA)、抑制性tRNA(Gly/UGA)、抑制性tRNA(Cys/UGA)、抑制性tRNA(Leu/UGA)、抑制性tRNA(Ser/UGA)；Ocher抑制性tRNA：抑制性tRNA(Gln/UAA)、抑制性tRNA(Tyr/UAA)、抑制性tRNA(Lys/UAA)、抑制性tRNA(Glu/UAA)、抑制性tRNA(Leu/UAA)、抑制性tRNA(Ser/UAA)。
(2)SOE PCR引物及点突变引物设计
根据以确定的19中抑制性tRNA序列合成19种5’端连接有7sk启动子的19种抑制性tRNA其中3种抑制性tRNA，抑制性tRNA(Gln/UAG)、抑制性tRNA(Tyr/UAG)、抑制性tRNA(Lys/UAG)由序列全合成得到，其余13种由SOE PCR法合成抑制性tRNA并在5‘端连接7sk启动子，3种由得到的抑制性tRNA点突变获得。
表1 7sk启动子序列
表2 SOE PCR引物序列
表3 抑制性tRNA点突变引物设计
| 点突变引物名称 | 引物序列(5’-3’方向) |
| 7sk-Gln-UGA for | ctcggatcgctggatttgaagtccagagtgctaac |
| 7sk-Gln-UGA rev | gttagcactctggacttcaaatccagcgatccgag |
| 7sk-Tyr-UAA for | gcgacctaaggatctaaagtcctccgctctacc |
| 7sk-Tyr-UAA rev | ggtagagcggaggactttagatccttaggtcgc |
| 7sk-Trp-UAG-for | gcaacggcagcgcgtctgactctagatcagaaggt |
| UAG-Trp-rev | accttctgatctagagtcagacgcgctgccgttgc |
(3)抑制性tRNA连接至Bjmu载体
在得到抑制性tRNA的基础上，用BamHI和Bgl II双酶切抑制性
tRNA，BamHI单酶切Bjmu载体，将酶切产物连接，得到连有7sk-抑制性ttRNA的Bjmu载体。
实施例2：利用双荧光素报告基因和点突变的GFP报告基因检测19种抑制性tRNA通读效率
(1)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：21。
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4 GFP突变引物列表
利用定点突变试剂盒(
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。
(2)293T细胞中分别转染不同抑制性tRNA质粒和双荧光素酶报告基因验证抑制性tRNA通读效率
将抑制性tRNA载体与和双荧光素报告基因pGL4-2luc-TAG；pGL4-2luc-TAA；pGL4-2luc-TGA质粒按表5的分组以1∶2比例混合，再与转染试剂megatrans 1.0按1∶3比例混合，共同加入293T细胞，6小时后换液，至细胞于37℃，5％CO2的孵箱中继续培养48小时后裂解细胞，向细胞裂解液加入荧光素酶底物，检测荧光读值。结果如图2b所示。加入抑制性tRNA后，可以得到全长有活性的突变型萤火虫荧光素酶蛋白。最终确定通读UAG、UAA、UGA效率最高的抑制性tRNA分别为Amber抑制性tRNA(Gln)；Ocher抑制性tRNA(Gln)；Opal抑制性tRNA(Arg)。
表5 双荧光素酶报告基因转染质粒分组
| 组别 | 质粒 |
| 1 | Bjmu-stRNAGln-UAG和pGL4-2luc-TAG |
| 2 | Bjmu-stRNATyr-UAG和pGL4-2luc-TAG |
| 3 | Bjmu-stRNALys-UAG和pGL4-2luc-TAG |
| 4 | Bjmu-stRNALeu-UAG和pGL4-2luc-TAG |
| 5 | Bjmu-stRNAGlu-UAG和pGL4-2luc-TAG |
| 6 | Bjmu-stRNATrp-UAG和pGL4-2luc-TAG |
| 7 | Bjmu-stRNAArg-UGA和pGL4-2luc-TGA |
| 8 | Bjmu-stRNAGln-UGA和pGL4-2luc-TGA |
| 9 | Bjmu-stRNATrp-UGA和pGL4-2luc-TGA |
| 10 | Bjmu-stRNAGly-UGA和pGL4-2luc-TGA |
| 11 | Bjmu-stRNACys-UGA和pGL4-2luc-TGA |
| 12 | Bjmu-stRNALeu-UGA和pGL4-2luc-TGA |
| 13 | Bjmu-stRNASer-UGA和pGL4-2luc-TGA |
| 14 | Bjmu-stRNAGln-UAA和pGL4-2luc-TAA |
| 15 | Bjmu-stRNATyr-UAA和pGL4-2luc-TAA |
| 16 | Bjmu-stRNALys-UAA和pGL4-2luc-TAA |
| 17 | Bjmu-stRNAGlu-UAA和pGL4-2luc-TAA |
| 18 | Bjmu-stRNALeu-UAA和pGL4-2luc-TAA |
| 19 | Bjmu-stRNASer-UAA和pGL4-2luc-TAA |
(3)293T细胞中分别转染19种抑制性tRNA和pcDNA3.1-GFP质粒验证抑制性tRNA通读效率
将实施例2的步骤1获得的pcDNA3.1-GFP-39TXX，以及实施例1的步骤3的19种抑制性tRNA质粒按表6的分组按照实施例2步骤2所述转染方式转染至293T细胞，48小时后后用荧光显微镜观察绿色荧光，结果如图2c所示。最终进一步验证通读UAG、UAA、UGA效率最高的抑制性tRNA分别为Amber抑制性tRNA(Gln)；Ocher抑制性tRNA(Gln)；Opal抑制性tRNA(Arg)。
表6 GFP报告基因转染质粒分组
| 组别 | 质粒 |
| 1 | Bjmu-stRNAGln-UAG和pcDNA3.1-GFP-39TAG |
| 2 | Bjmu-stRNATyr-UAG和pcDNA3.1-GFP-39TAG |
| 3 | Bjmu-stRNALys-UAG和pcDNA3.1-GFP-39TAG |
| 4 | Bjmu-stRNALeu-UAG和pcDNA3.1-GFP-39TAG |
| 5 | Bjmu-stRNAGlu-UAG和pcDNA3.1-GFP-39TAG |
| 6 | Bjmu-stRNATrp-UAG和pcDNA3.1-GFP-39TAG |
| 7 | Bjmu-stRNAArg-UGA和pcDNA3.1-GFP-39TGA |
| 8 | Bjmu-stRNAGln-UGA和pcDNA3.1-GFP-39TGA |
| 9 | Bjmu-stRNATrp-UGA和pcDNA3.1-GFP-39TGA |
| 10 | Bjmu-stRNAGly-UGA和pcDNA3.1-GFP-39TGA |
| 11 | Bjmu-stRNACys-UGA和pcDNA3.1-GFP-39TGA |
| 12 | Bjmu-stRNALeu-UGA和pcDNA3.1-GFP-39TGA |
| 13 | Bjmu-stRNASer-UGA和pcDNA3.1-GFP-39TGA |
| 14 | Bjmu-stRNAGln-UAA和pcDNA3.1-GFP-39TAA |
| 15 | Bjmu-stRNATyr-UAA和pcDNA3.1-GFP-39TAA |
| 16 | Bjmu-stRNALys-UAA和pcDNA3.1-GFP-39TAA |
| 17 | Bjmu-stRNAGlu-UAA和pcDNA3.1-GFP-39TAA |
| 18 | Bjmu-stRNALeu-UAA和pcDNA3.1-GFP-39TAA |
| 19 | Bjmu-stRNASer-UAA和pcDNA3.1-GFP-39TAA |
实例3：293T细胞系中通读疾病蛋白Dystrophin
(1)构建含有提前终止密码子UAG、UAA、UGA的Dp71b突变质粒
Dystrophin蛋白的同源异构体Dp71b序列如SEQ ID NO：23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Dp71b3115TAG，突变为c.9346C＞T，含有提前终止密码子UAA的Dp71b3216TAA，突变为c.9651C＞A；含有提前终止密码子UGA的Dp71b3112TGA，突变为c.9337C＞T，经测序验证突变成功。
(2)293T细胞系中通读疾病蛋白Dystrophin
将实施例3的步骤1获得的Dp71b3115TAG，Dp71b3216TAA，Dp71b3112TGA质粒与对应的抑制性tRNA按照实施例2步骤2所述转染方式转染至293T细胞，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3a。证明抑制性tRNA能够通读不同类型提前终止密码子，恢复疾病蛋白的表达。
实施例4：抑制性tRNA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Bjmu-stRNAGln-UAG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，置细胞于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3b和3c。验证抑制性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。
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。
Claims (15)
- 快速构建抑制性tRNA的方法，其特征在于所述方法包括下列步骤：(1)设计覆盖全部抑制性tRNA并部分互补的上下游引物，同时设计7sk基因PCR引物，7sk基因PCR下游引物能同时与7sk和抑制性tRNA互补；(2)第一步PCR分别使用对应的引物合成并扩增抑制性tRNA及扩增7sk启动子序列；(3)第二步PCR连接抑制性tRNA与7sk启动子，并扩增连接产物，最终得到带有7sk启动子的抑制性tRNA；其特征在于所述抑制性tRNA由tRNA反密码子环突变得到。
- 根据权利要求1的方法，其特征在于所述7sk启动子序列为表1所示的序列。
- 根据权利要求1的方法，其特征在于所述引物选自表2所示的序列。
- 筛选抑制性tRNA的方法，其包括：(1)确定人类遗传性疾病中19种无义突变频率较高的氨基酸密码子，改变识别这19种密码子的相应tRNA反密码子环碱基；(2)构建无义突变抑制性tRNA，并在抑制性tRNA 5’端连接7sk启动子；(3)将抑制性tRNA与含有提前终止密码子的突变蛋白基因转染至宿主细胞，在宿主细胞中恢复突变蛋白正常表达；(4)比较不同抑制性tRNA促进通读效率，比较得到通读效率高的抑制性tRNA。
- 根据权利要求1-4任一项的方法，其特征在于，所述方法得到的抑制性tRNA选自SEQ ID NO：1-19所示的抑制性tRNA。
- 根据权利要求4的方法，其特征在于，所述突变蛋白选自双荧光素酶报告基因蛋白、GFP蛋白、dystrophin蛋白、STK11蛋白和EPHB2蛋白。
- 权利要求1-6任一项的方法得到的抑制性tRNA。
- 权利要求7的抑制性tRNA，其特征在于，所述抑制性tRNA 选自序列如SEQ ID NO：1-19所示的抑制性tRNA。
- 包含权利要求7或8的tRNA的质粒、载体或试剂盒。
- 权利要求9的试剂盒，其特征在于，其包含序列如SEQ ID NO：1-19所示的抑制性tRNA。
- 根据权利要求10的试剂盒，其特征在于，其包含Amber抑制性tRNA(Gln)对应SEQ ID NO：1；Ocher抑制性tRNA(Gln)对应SEQ ID NO：14；Opal抑制性tRNA(Arg)对应SEQ ID NO：7。
- 权利要求7或8的tRNA或权利要求9的质粒、载体或权利要求9-11任一项的试剂盒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。
- 根据权利要求12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。
- 评价抑制性tRNA通读无义突变的效率的方法，其包括：(1)将报告基因进行点突变，得到分别含有UAG、UAA、UGA三种提前终止密码子的突变体，并连接适当的载体；(2)将步骤(1)获得的包含突变报告基因的载体与不同抑制性tRNA分别共转染至宿主细胞之中；(3)检测报告基因，根据报告基因检测结果确定抑制性tRNA通读效率。
- 恢复单基因遗传病的致病基因和肿瘤细胞内的肿瘤抑制基因无义突变体的截短蛋白表达的方法，包括将权利要求7或8的tRNA或权利要求9的质粒、载体导入含有无义突变体蛋白的细胞或生物体内，优选地，使用权利要求9-11任一项的试剂盒进行。
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