A kind of transmembrane fluorescent protein and its coding gene and application
A fluorescent protein and gene-encoding technology, applied in the direction of microbial-based methods, using vectors to introduce foreign genetic material, peptides, etc., can solve the problems of time-consuming, high protein cost, etc., and achieve the effect of reducing toxicity and preventing degradation
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Embodiment 1
[0055] This embodiment provides an artificially modified green fluorescent protein, which is selected from eight modified green fluorescent proteins. These proteins are chemically synthesized polypeptides. The specific sequence of an artificially modified green fluorescent protein is shown as sequence 1 in the sequence list. Compared with the unmodified or other molecularly modified green fluorescent protein, only the artificially modified green fluorescent protein provided by the present invention can efficiently penetrate the cell membrane of insect cells.
[0056] In this example, 8 artificially modified green fluorescent proteins and natural sequence green fluorescent proteins were obtained by chemical synthesis of polypeptides, and the above proteins were compared for their membrane-penetrating ability to insect cells. The specific analysis methods and results are as follows.
[0057] Experimental method 1: Insect cell SF9 was cultured to 90% growth density in insect cel...
Embodiment 2
[0060] This embodiment provides an optimized artificially synthesized artificially modified green fluorescent protein gene with a 6×His tag at the C-terminal, and the specific sequence is shown as sequence 2 in the sequence listing. The optimized DNA sequences were compared by NCBI, and there was no obvious similarity.
[0061] The optimized DNA with a 6×His tag at the C-terminal was connected to the Pichia pastoris secretory expression vector pPICZαA to obtain recombinant vectors, and then the recombinant vectors were transformed into In Pichia pastoris host strain X-33, after transformation, use YPD plates containing 100 µg / mL Zeocin antibiotics to screen, use PCR to verify the transformants, and inoculate the Pichia transformants after PCR verification into the YPD plates containing different concentrations of Zeocin antibiotics were screened to obtain highly resistant Pichia transformants containing a modified green fluorescent protein gene, and then small amounts of BMMY ...
Embodiment 3
[0063] This embodiment provides a method for preparing protein, which specifically includes the following steps:
[0064] S1: Construction of expression vector and transformation: The artificially synthesized DNA with a 6×His tag at the C-terminal of Example 1 was connected to the secretory expression vector pPICZαA of Pichia pastoris to obtain the recombinant vector pPICZαA-RGFP, and the vector construction was as follows figure 2 as shown, figure 2 A schematic diagram of the construction of the eukaryotic expression vector pPICZαA-RGFP in the embodiment of the present invention. The main vector construction steps are preferably as follows:
[0065] (1) Use Xho Ⅰ and Xba Ⅰ to double digest the plasmid containing a modified green fluorescent protein gene to obtain the target fragment. The reaction system is as follows (the endonuclease and buffer used are purchased from Dalian TAKARA Company):
[0066] Plasmid containing the synthetic AaIT gene 15 μL
[0067] 5 μL of 10×M...
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