Preparation method and application of human ptx3 recombinant protein
A recombinant protein and human technology, which is applied in the field of preparation of human recombinant protein, can solve the problem that the technical level of CHO-K1 cells expressing foreign genes cannot meet the requirements of the development and production of biopharmaceuticals, low production efficiency of recombinant CHO-K1 cells, sugar Insufficient stability of ylated expression products, etc., to achieve the effects of increasing the survival time, high RNA extraction yield, and increasing the success rate
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[0055] A method for preparing human PTX3 recombinant protein of the present invention, said method comprising the following steps:
[0056] S1. Extract mRNA carrying PTX3 protein translation information from human primary epithelial cells, design specific primers, use the mRNA as a template, synthesize cDNA, and introduce restriction enzyme sites at both ends of the cDNA BamH I and Bg1 II;
[0057] S2, exposing the sticky ends of BamH I and Bg1 II on the eukaryotic cell expression vector pSG5 with endonuclease, and cloning the above-mentioned cDNA with the ends of BamH I and Bg1 II into the expression vector to construct pSG5 / PTX3 vector , inserting a neomycin resistance gene into the pSG5 / PTX3 vector to construct a recombinant vector pSG5 / neo- / PTX3;
[0058] The neomycin resistance gene was inserted into the expression vector pSG5 / PTX3 to form a pSG5 / PTX3 / neo recombinant expression vector, and the stable transfected CHO-K1 cells were selected by neomycin resistance. Only ce...
Embodiment 1
[0084] Example 1: Construction of vector pSG5 / PTX3 / neo
[0085] 1. Construction of pSG5 / PTX3
[0086] 1.1 Preparation of PTX3 gene fragment
[0087] The TRIzol method was used to extract total RNA from primary human epithelial cells. The total RNA contains mRNA carrying human PTX3 protein information; according to the human PTX3 mRNA sequence published on the GenBank website, specific primers are designed, and the PTX3 mRNA in the total RNA is used as a template to synthesize cDNA. And respectively introduce restriction enzyme sites BamH I and Bg1 II at both ends of the cDNA, the specific steps are as follows:
[0088] Reverse transcription polymerase chain reaction (RT-PCR)
[0089] A pair of primers (P1 and P2) were designed, the restriction enzyme cutting site BamH I was introduced into the upstream primer, and the restriction enzyme cutting site Bg1 II was introduced into the downstream primer. Under the action of reverse transcriptase, the first strand of cDNA (SEQ ID...
Embodiment 2
[0110] Embodiment 2: the method for purifying recombinant human PTX3 protein:
[0111] The culture supernatant from the fermentor was loaded onto a Q-Sepharose™ Fast Flow (GE Healthcare, UK) packed column. The remaining material is eluted using a nonlinear gradient. The eluate containing PTX3 was directly applied to ceramic hydroxyapatite (BioRad, Hercules, CA, USA) and packed into a column. The remaining material is eluted in a non-linear fashion by increasing the phosphate concentration. The eluate containing PTX3 was concentrated and buffer exchanged on an ultrafiltration membrane (Pellicon-Biomax 100, Millipore) followed by purification by size exclusion chromatography on a Biosep-SEC-S4000 (Phenomenex). Finally, the PTX3 eluate was concentrated with an ultrafiltration membrane (Pellicon-Biomax 100, Millipore).
[0112]After Western blot (WB) result analysis, after purification, the recovery rate of PTX3 recombinant protein reaches 69%, and the purity reaches 95%, as ...
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