Expression vector for bimolecular fluorescence complementation) research and application thereof
An expression vector and bimolecular technology, which can be used in the introduction of foreign genetic material, material inspection products, recombinant DNA technology, etc. using vectors. It can solve problems such as high price and achieve high flexibility.
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Embodiment 1
[0050] Example 1 Construction of a new expression vector pBiFC-GG for bimolecular fluorescence complementation research
[0051] 1) Transform the multiple cloning sites of the traditional BiFC vectors pSAT-cEYFP-C1, pSAT-nEYFP-C1, pSAT-cEYFP-N1 and pSAT-nEYFP-N1, respectively introduce the GG cloning frame structure, and transform the vectors respectively Named pSAT-cC1-GG, pSAT-nC1-GG, pSAT-cN1-GG and pSAT-nN1-GG;
[0052] Wherein, the GG cloning frame structure includes BsaI restriction site, SfiI restriction site, ccdB gene, SfiI restriction site, BsaI restriction site connected in sequence, the nucleosides of the GG cloning frame structure The acid sequence is shown in SEQUENCE NO.1. Schematic diagram of the structure of the GG cloning cassette figure 1 As shown, the wavy line represents the enzyme cleavage site.
[0053] Specifically, the primer pair CccdBF: gagacctagttctgggccaggatggccattctcgactaagttggcag and CccdBR: aaccacctgagaccgaaggcccagatggccctgtgtataagggagcctg we...
Embodiment 2
[0063] Example 2 Using pBiFC-GG-N1 to study the interaction between papaya eIF4E and PRSV-Vpg
[0064] The inventor's laboratory team has used yeast two-hybrid technology to find that papaya eIF4E interacts with papaya ringspot virus PRSV-Vp, so these two proteins are selected to verify the reliability of the pBiFC-GG-N1 vector in the present invention sex.
[0065] 1. Amplification of papaya eIF4E and PRSV-Vpg genes
[0066] 设计两对扩增番木瓜eIF4E基因的引物,Golden-4EF:acaggtctcagtctatggtagtagaaggaaccc和Golden-4ER:acaggtctcacacctaccgagtagcgattcttag,以及Gibson-4EF:acctaggttctgggccaggaatggtagtagaaggaaccc和Gibson-4ER:cctgagaccgaaggcccagataccgagtagcgattcttag,其中引物对Golden-4E用于GoldenGate克隆,引物对 Gibson-4E was used for closed loop Gibson splice clones.
[0067]设计两对扩增番木瓜环斑病毒PRSV-Vpg基因的引物,Golden-VpgF:acaggtctcatggcatgggtttctccgcgcgacag和Golden-VpgR:acaggtctcactgattcatggtgaacagattttgtt,以及Gibson-VpgF:gagaccatggagggccttcaatgggtttctccgcgcgacag和Gibson-VpgR:cctgaagagaccggcccactttcatggtgaacagattttgtt,其中引物对Golden...
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