Novel purification and renaturation method and antitumor application of recombinant endostatin
A column purification and vascular endothelium technology, which is applied in the fields of genetic engineering and biomedicine, can solve the problems of low soluble protein yield, low yield, and limited clinical promotion and use of protein drugs.
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Embodiment 1
[0081] Embodiment 1, obtaining rhED inclusion body
[0082] The amino acid sequence of the designed recombinant rhED is shown in SEQ ID NO:2; its cDNA is shown in SEQ ID NO:1. Compared with the natural sequence, the ATG of this recombinant rhED is followed by "AGAGGATCC" encoding arginine, glycine and serine. "CACCATCATCATCATCAC" encoding 6×His was added upstream of the 3' TGA of the sequence.
[0083] Construction of pQE3 expression vector with rhED cDNA: the above rhED cDNA was obtained by gene synthesis, and inserted into the BamHI and HindIII sites of pQE3 (QIAGENUSA).
[0084] The pQE3 expression vector carrying rhED cDNA was transfected into Escherichia coli (E.ColiM15) to obtain rhED-producing bacteria, and the expression was induced with IPTG (0.25G / L), cultured at 37°C for 4 hours, and the bacteria were harvested by centrifugation at low temperature for 30 minutes and lysed. The recombinant rhED inclusion body was dissolved in 6M guanidine hydrochloride solution (0....
Embodiment 2
[0085] Example 2, optimization of purification and renaturation scheme of rhED
[0086] Scheme 1, scheme A of urea gradient solution dialysis-purification
[0087] The renaturation step of dialysis with urea gradient solution: diluent (0.05mol / Ltris, 2mmol / LEDTA, 3.5mol / L urea, 4mmol / L reduced glutathione (GSH), 0.4mmol / L oxidized glutathione Glycine (GSSG, pH8.0) dilute the inclusion body solution dissolved in guanidine hydrochloride to 0.3 mg / mL, dialyze with the dialysate A-G in Table 1 in sequence at 4°C, dialyze each dialysate once, each time for 24 hours, The ratio of the inner and outer fluids of the dialysis bag was 3:20, the inner fluid was 3.5M urea sodium acetate buffer, and the composition of the outer fluid (dialysis fluid) was shown in Table 1.
[0088] Table 1
[0089]
[0090] After refolding, centrifuge at 10000rpm for 15min, discard the precipitate, and the supernatant is the refolded rhED protein. Take OD 595 Draw a standard curve corresponding to the...
Embodiment 3
[0109] Example 3, rhED inhibits HUVEC cell growth and chicken embryo allantoic membrane angiogenesis inhibition
[0110] 1. rhED inhibits the growth of human umbilical vein endothelial cells (HUVEC)
[0111] Human umbilical vein endothelial cells (HUVEC) were purchased from ATCC and cultured in M199 basal medium containing 10% heat-inactivated fetal bovine serum and double antibodies. In the growth inhibition experiment, HUVEC was divided into 5×10 3 Each hole was inoculated on a 96-well plate with a volume of 100 μl, and cultured for 72 hours with the following culture solution (200 μl volume): (1), M199 basal culture solution+bFGF (5 ng / ml); (2), (1 )+0.1μg / mlrhED; (3), (1)+0.2μg / mlrhED; (4), (1)+0.5μg / mlrhED; (5), (1)+1μg / mlrhED; (6), ( 1) + 2 μg / mlrhED; (7), (1) + 5 μg / mlrhED; (8), (1) + 8 μg / mlrhED; (9), (1) + 16 μg / mlrhED. Then 20 μl MTT (5%) was added to each well, discarded after incubation for 4 hours, and shaken with 100 μl DMSO on a shaker for 15 minutes, and the...
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