Introduction of commonly used carrier components and application of virus vectors

1. Common vector classification:
2, commonly used carrier element introduction:
(1) initiators:
initiators are located in the structure
gene
5' side upstream of the
DNA
sequence, can activate the RNA polymerase, so that it is accurately combined with the template DNA and has the specificity of transcription starting.
starters are mainly divided into broad-spectrum initiaters and specific initiaters. A broad-spectrum initiater is a
-based
widely expressed in various
tissues and cells, and a specific initiater is a initiater expressed in a specific tissue or cell type. Common broad-spectrum initiaters are CAG, CMV, EF1a, PGK, etc., and also contain type III launchers U6 and H1 launchers that initiate RNA; n, projection neuron-specific initiation CamKIIa, astrocyte-specific initiation GfaABC1D, myocardial-specific initiation cTNT, hepatic essential cell-specific initiation TBG, etc.
(2) Fluorescence:
non-fusion expression of encoded proteins, conventionally recommended target genes and fluorescent proteins, and the efficiency of infection is observed through fluorescence. If the location of the target gene needs to be marked with fluorescence, such as subcellular positioning and other experiments, the fusion expression of the target gene and fluorescent protein can be considered. Because proteins have their own spatial structure, if you choose fusion expression, the two proteins may affect each other.
common structures for fusion expression:
non-fusion expression common structures:
(3) Resistance:
A Common primary nucleation resistance: Amp, Kan.
B commonly used nuclear resistance: Puro, Blasticidin (BSD, BSR), Neo (G418), Zeo, Hygro, where Puro and BSR are the more commonly used hyal nuclear resistance. True nuclear resistance is often used to stabilize plant screening and other processes, the specific use of
resistance screening concentration can be confirmed by the empty cell resistance pre-experiment, concentration gradient can be based on the
antibiotics
instructions recommended concentration range settings.
(4) Protein Label:
Protein Tag: A
peptide
or protein expressed in fusion with the destination protein for the expression, detection, dema and purification of the end protein. Common labels: 3xFLAG, 6xHis, HA, Myc, OLLAS, GST, etc. Attention should be paid to whether the purpose protein has functional peptides such as signal peptides, pre-peptides, etc., protein labels need to fuse one end of the functional peptide.
(5) Other:
Self-cut Peptide 2A (self-cleaving 2A peptide, 2A): Average length 18-22
amino acids
The genes of 2A upstream and downstream are transscribed at the same time, while the translated peptide segment is disconnected at the last proline of 2A, resulting in an upstream protein fused with a 2A peptide tail and a downstream protein with a proline at the N end, respectively.
: 2A peptide structure is
short and the expression balance of upstream and downstream genes is good;internal ribosome entry site (
IR
ES): The IRES element has the ability to independently collect ribosomes without relying on the hat structure, which in turn can initiate the translation of downstream genes. Two genes upstream and downstream of the IRES element are trinscribed at the same time, translated independently, and two separate proteins are eventually obtained.
Advantages:
independent translation, which in turn can express the complete purpose protein;
Disadvantages:
IRES structure is large, its application is often limited by carrier capacity, and IRES on the downstream gene translation starting capacity is relatively weak, so often in order to ensure the expression of the destination gene, fluorescent protein in the lower IRES, may lead to weak fluorescence.。 3. Comparison and application of different virus vectors: