Nucleic acid sequence for potentiating the expression of useful gene and method therefor
A nucleic acid sequence and gene technology, which is applied in the field of nucleic acid sequences and methods for enhancing the expression of useful genes, and can solve problems such as inefficient gene expression
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Embodiment 1
[0146] Embodiment 1: the preparation of HCV nucleic acid sequence fragment
[0147] The cDNA sequence of each IRES region containing the 5' untranslated region of HCV was prepared as a nucleic acid sequence for enhancing expression of useful genes. mRNA was extracted from serum of HCV patients according to standard methods, and cDNA was synthesized by reverse transcriptase (GIBCO BRL) reaction. Using the cDNA as a template, use the primers of the following sequence (with a HindIII recognition site added at the 5' end; SEQ ID NO: 2-6) to perform PCR to amplify specific IRES active fragments, including HCV 5' untranslated Region (SEQ ID NO: 1; nucleotides 1-341) and part of the core protein coding region (SEQ ID NO: 1; nucleotides 342-713):
[0148] 5'HindHCV001: 5'-ccc aag ctt gcc agc ccc ctg atg ggg gcg a-3' (SEQ ID NO: 2)
[0149] 5'HindHCV180: 5'-ccc aag ctt ctg gca att ccg gtg tac tca c-3' (SEQ ID NO: 3)
[0150] 5'HindHCV181: 5'-ccc aag ctt gac gac cgg gtc ctt tct tg-3'...
Embodiment 2
[0154] Example 2: Transient transfection
[0155] Two vectors were obtained respectively, i.e. pGL2B vector (i.e., pGL2 base plasmid (Promega) containing SV40 poly A signal and cDNA sequence encoding firefly luciferase, without promoter, enhancer and nucleic acid sequence for enhanced expression) and pGL2C vector (i.e., containing firefly luciferase, SV40 poly A signal and SV40 early promoter / enhancer, but without the nucleotide sequence pGL2 control plasmid (Promega) for enhanced expression see Figure 2(a)), and pGL2B UTR5'-3' was prepared as follows (HCV 1-341 obtained in Example 1 was inserted into the HindIII site of pGL2B in the 5'-3' direction, see FIG. 2(b)).
[0156] Among the HCV cDNA fragments with the HindIII recognition site obtained by the above PCR, HCV1-341 was selected to be digested with HindIII at 37°C overnight, and the pGL2B vector was also digested with HindIII, and then alkaline phosphatase (Boehringer Mannheim) Treat for 30 minutes to avoid intramolecul...
Embodiment 3
[0163] Example 3: Transient transfection
[0164] In order to construct the useful gene expression vector of the present invention, use a similar method to prepare pGL2B UTR5'-3', and use HindIII to prepare pGL2C UTR5'-3', that is, insert HCV1-341 at the HindIII site in the 5'-3' direction pGL2C (see Figure 2C).
[0165] According to the method of Example 2, COS1 cells were transfected with 5 μg of vector DNA, namely pGL2C or pGL2C UTR5'-3', and then the luciferase activity was measured.
[0166] The results are shown in Table 2.
[0167] carrier
[0168] The above data clearly show that when HCV1-341 is inserted between the luciferase gene and the promoter sequence in the 5'-3' direction in the expression vector, the luciferase activity increases. This shows that the 5'untranslated region of HCV gene has the effect of enhancing gene expression.
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