Recombinant human endostatin protein with different amino acid structures, method for preparing recombinant human endostatin protein and application thereof
A technology of endostatin and amino acids, applied in the field of genetic engineering, can solve the problems of single-drug administration and high probability of antibody production
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Embodiment 1
[0050] The genetically engineered recombinant protein of embodiment 1 recombinant plasmid pET9c-En1 and its preparation method
[0051] This patent shows the comparison of the amino acid sequence and nucleotide sequence encoding prokaryotic expression endostatin 1 with the endostatin that has been approved for invention patent or approved for clinical research as a drug.
[0052] The sequence of PCR primers in the genetically engineered recombinant protein of the recombinant plasmid pET9c-En1 and its preparation method is as follows:
[0053] Primer (1): ATTCATATGCGCCGCCGCCGCCGCCGCCGCCGCCGCCACAGCCACCGCGACTTCCAG (SEQ ID NO.8)
[0054] Primer (2): GCCGGATCCCTACTTGGAGGCAGTCATGAAGCT (SEQ ID NO.9)
[0055] The primers for the PCR reaction were synthesized by Shanghai Sangon Company, using the endostatin nucleotide sequence in Xu Genxing Zl97107112.8 invention patent as shown in SEQ ID NO.7 as a template, adding the above two primers and conventional PCR reaction reagents, PCR reac...
Embodiment 2
[0058] The genetically engineered recombinant protein of embodiment 2 recombinant plasmid pET9c-En2 and its preparation method
[0059] This patent shows the comparison of the amino acid sequence and nucleotide sequence encoding the prokaryotic expression recombinant human endostatin protein 2 with Example 1 and the endostatin that has been approved for invention patents or approved for clinical research as a drug.
[0060] The sequence of PCR primers in the genetically engineered recombinant protein of the recombinant plasmid pET9c-En2 and its preparation method is as follows:
[0061] Primer (3) CATATGCGCCGCCGCCGCCGCCGCCGCCGCCGCCACAGCCACCGCGACTTCC (SEQ ID NO.10)
[0062] Primer (4) GCGGCATGCGGGGCGATCGCGGGGACTTCCAGTGCTTCC (SEQ ID NO.11)
[0063] Primer (5) GGAAGCACTGGAAGTCCCCGCGATCGCCCCGCATGCCGC (SEQ ID NO.12)
[0064] Primer (6) GTGGCATGGCTCGGACGCAAACGGGCGCAGGCTG (SEQ ID NO.13)
[0065] Primer (7) CAGCCTGCGCCCGTTTGCGTCCGAGCCATGCCAC (SEQ ID NO.14)
[0066] The primers of ...
Embodiment 3
[0067] Embodiment 3 Genetic engineering recombinant protein of recombinant plasmid pMAL-c2-En1 and pMAL-c2-En2 and preparation method thereof
[0068] Primer (8) CCGGAATTCCGCCGCCGCCGCCGCCGCCGCCGCCGCCAC (SEQ ID NO.15)
[0069] According to the method of Example 1, the endostatin nucleotide sequence of pET9c-En1 and the endostatin nucleotide sequence of pET9c-En2 were respectively used as templates, and primers (8) and primers (2) were respectively added to conventional PCR reactions The reagents were used for PCR, and the obtained PCR product and the purified pMAL-c2 vector were respectively digested with EcoRI and BamHI, and cloned according to the method in Example 1 to obtain pMAL-c2-En1 and pMAL-c2-En2 plasmids. Transform the empty vector pMAL-c2 and the recombinant plasmids pMAL-c2-En1 and pMAL-c2-En2 into E.coliBL21(DE3) respectively, inoculate them in LB medium containing 100 μg / mL penicillin, and wait for them to grow to OD 600When it was 0.4-0.6, IPTG (final concentra...
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