Animal cell bicistronic high-efficiency expression vector
A bicistronic and expression carrier technology, applied in the biological field, can solve the problems of low expression efficiency, high cost, and complex structure of foreign protein in animal cells, so as to increase the expression amount and level of foreign protein and reduce production cost effect
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Embodiment 1
[0054] Example 1 Construction of SV40 late promoter
[0055] (1) Amplification of SV40 late promoter
[0056] ① Design primers F01 / R01 with the SV40 late promoter sequence in the SV40 genome (NCBI accession number: NC_001669) as a reference, and pSVL plasmid (Amersham) as a template for PCR amplification. The PCR reaction conditions are shown in Table 1.
[0057] F01 ACGTCAATGGGAGTTTGTTTTAAGCTTTTTGCAAAAGCCTAGGCCTC (SEQ ID NO: 5)
[0058] R01 GGTGGCTAGCGGCCTGAAATAACCTCTGAAAGAGGAACTTG (SEQ ID NO: 6)
[0059] Table 1 PCR reaction conditions
[0060]
[0061] ②Design primers F012 / R01 with the SV40 late promoter sequence in the SV40 genome as a reference, and pSVL plasmid (Amersham) as a template for PCR amplification. The PCR reaction conditions are shown in Table 1.
[0062] F012 ATTGACTAGTAAGCTTTTTGCAAAAGCCTAGGCCTCCAAA (SEQ ID NO: 7)
[0063] The resulting PCR product was ligated with pUC57 (purchased from Fermentas) treated with SmaI, and sequenced and identified.
[0064] (2) CMV enhancer a...
Embodiment 2
[0070] Example 2 Synthesis of SV40 late polyA signal sequence
[0071] (1) Amplification of SV40 late polyA signal
[0072] Using pSVL as a template, the SV40 late polyA signal sequence in the pSVL sequence was used as a reference to design primers F03 / R03. The PCR reaction conditions are shown in Table 1.
[0073] F03: TATATCTAGACAGACATGATAAGATACATTGATGAGTTTGGACAAACCAC (SEQ ID NO: 10)
[0074] R03: GTCGACGGTATACTACCACATTTGTAGAGG (SEQ ID NO: 11)
[0075] The resulting PCR product was connected to pUC57 treated with SmaI, and sequenced for identification.
[0076] (2) Synthesis of SV40 late polyA signal of the present invention
[0077] Taking the SV40 late polyA signal sequence in the SV40 genome as a reference, a repetitive sequence was artificially added, which is a repetitive sequence on the SV40 late polyA signal region. Design primers as follows:
[0078] 1 ACAAATCTAGACAGACATGATAAGATACATTGAT (SEQ ID NO: 12)
[0079] 2 AGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTCT (SEQ ID NO: 13)
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Embodiment 3
[0091] Example 3 Construction of expression vector
[0092] The product CMV enhancer / SV40 late promoter in Example 1 (3) and pIRESneo3 plasmid were subjected to SpeI / NheI double enzyme digestion and connection to obtain plasmid pCSe.
[0093] The SV40 late polyA signal of Example 2 (1) was ligated with the pCSe plasmid by XbaI / BST1107I double enzyme digestion, and the resulting plasmid was named pCSS.
[0094] The pCSS and the SV40 late promoter of Example 1 (1) ② were subjected to SpeI / NheI double enzyme digestion and connection, and the resulting plasmid was named pSVS.
[0095] The SV40 late polyA signal of the present invention of Example 2 (2) was ligated with pCSe by XbaI / BST1107I double enzyme digestion, and the obtained plasmid was named pCSL.
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