Recombined protein of anti tumour, encoded gene and application
A recombinant protein, anti-tumor technology, applied in the direction of recombinant DNA technology, the introduction of foreign genetic material, hybrid peptides using vectors, etc., can solve the problems of inability to kill tumor cells, reduced blood picture of patients, bleeding, etc., to improve curative effect, reduce The effect of dosage and simple operation
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Embodiment 1
[0048] Example 1. In vitro expression of anti-tumor recombinant protein in Pichia pastoris
[0049] 1. Synthesis of anti-tumor recombinant protein gene
[0050] Artificially synthesized the full-length sequence of sequence 13 (the gene encoding cardiac troponin I) in the sequence list, and added NdeI and NotI restriction sites at the 5' end to obtain the sequence AFF1; the artificially synthesized sequence The 1-285 base sequence of sequence 7 in the list, and add NdeI restriction site at its 5' end, 3' end plus 15 bases complementary to the 5' end of gene sequence 13, then this The sequence is connected with the sequence AFF1 to obtain the sequence AFF2; bases 631-858 of the sequence 8 in the sequence list are artificially synthesized, and a NotI restriction site is added to the 3' end, and a 3' to the gene sequence 13 is added to the 5' end 15 bases complementary to each other, and then connect this sequence with the sequence AFF1 to obtain the sequence AFF3.
[0051] 2. I...
Embodiment 2
[0060] Example 2, Escherichia coli in vitro expression of anti-tumor recombinant protein
[0061] 1. Synthesis of artificial gene: artificially synthesize the full-length sequence of sequence 14 (the coding gene of cardiac troponin I) in the sequence table, and add NdeI and NotI restriction sites at its 5' end and 3' end, The sequence AFF4 is obtained; the 1-285 base sequence of sequence 9 in the sequence list is artificially synthesized, and an NdeI restriction site is added to its 5' end, and a 15 complementary to the 5' end of the gene sequence 14 is added to the 3' end. bases, and then connect this sequence with the sequence AFF4 to obtain the sequence AFF5; artificially synthesize the 631-858 bases of the sequence 10 in the sequence list, and add a NotI restriction site at its 3' end, and add a NotI restriction site at its 5' end 15 bases complementary to the 3' end of the gene sequence 14, and then this sequence was connected with the sequence AFF4 to obtain the sequence...
Embodiment 3
[0070] Example 3, in vivo stability experiment of Escherichia coli in vitro expression purified liquid of anti-tumor recombinant protein:
[0071] Get 24 Kunming mice, divide into phosphate buffer saline and expression purification group, subcutaneously inject phosphate buffer saline and the expression purification solution obtained in Example 2 respectively 2.0mg protein / kg, inject 8 hours, take blood from fundus vein , the serum was separated, and the serum anti-tumor recombinant protein concentration was determined by conventional enzyme-linked immunosorbent assay. Results The concentrations of serum anti-tumor recombinant protein in phosphate buffer solution and expression purification solution sequence AAF4, AAF5 and AAF6 were 0.52+0.08ng / ml, 3.55+0.12ng / ml, 8.22+1.08ng / ml and 9.31+1.62ng / ml, respectively. ml, indicating that fusion with troponin C can increase its stability in vivo and delay degradation.
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