Preparation method of recombinant baculovirus for expressing human transferrin receptor

Abstract

The invention discloses a preparation method of a recombinant baculovirus for expressing a human transferrin receptor. The method comprises the following steps: replacing a transmembrane region of a human transferrin receptor with a signal peptide and a transmembrane region amino acid sequence of influenza A virus neuraminidase, and optimizing a target gene to synthesize a gene sequence; the gene sequence is digested with EcoRI and NotI to obtain a target fragment with enzyme digestion sites, the insect cell expression vector pFastBac1 is digested with EcoRI and NotI, the digested target fragment and linearized vector are to purified and recovered, and a ligation reaction is performed through T4DNA ligase to obtain a ligation product; the ligation product is transformed into DH10Bac competent cells, and a recombinant baculovirus shuttle vector reBacmid-hTfR is obtained through resistant blue-white spot screening and separation; and transfecting the reBacmid-hTfR into an Sf9 insect cell, and carrying out cell subculture for three times, so as to obtain the recombinant baculovirus. The recombinant baculovirus can express hTfR on the surface of a cell membrane in the replication process, the antigenicity and solubility are maintained, and can be used for subsequent development of antibodies and biological functions.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a preparation method of a recombinant baculovirus expressing human transferrin receptor. Background technique [0002] Human transferrin receptor (hTfR), also known as CD71 molecule, is a transmembrane glycoprotein widely distributed in human tissues or cells, and its function is through the interaction with transferrin (Tf) Specific binding to participate in and mediate the absorption of iron plays an important role in the process of cell growth, proliferation and differentiation. The human transferrin receptor gene is located on chromosome 3 (3q26), with a total genome length of about 31kb, including 19 exons and 18 introns, and the mRNA formed after transcription is about 5kb in length, of which the Exon 1 is not involved in protein coding, and exon 2 to 19 encodes a protein, which encodes a monomer containing 760 amino acid residues. The human transferrin recepto...

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Problems solved by technology

[0003] The in vitro gene expression system includes prokaryotic expression system and eukaryotic expression system. The prokaryotic expression system mainly uses Escherichia coli as a bioreactor to obtain recombinant proteins. Its advantages are simple operation, short cycle and low cost, but its shortcomings cannot be ignored. For example, due to the lack of corresponding modification enzymes in E. coli, the corresponding post-transcriptional processing and modification of the expression product cannot be performed, and it is easy to produce inclusion bodies lacking the natural conformation

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