Anti-tumor nucleic acid and polypeptide and application thereof
An anti-tumor activity, tumor technology, applied in the field of genetic engineering, can solve the problem of ineffective anti-tumor drugs
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Embodiment 1
[0096] Construction of embodiment 1 expression plasmid
[0097] On the basis of the expression vector pET28a (Novagen, Inc.), an HA tag to facilitate immunohistochemical experiments and a TAT signal peptide to facilitate the expressed polypeptide to pass through the cell membrane were inserted.
[0098] First design a connection segment. To generate the ligated fragment, 4 sets of oligonucleotides were first synthesized and ligated end-to-end (see figure 2 ). The specific connection steps include: mixing 4 groups of oligonucleotide chains in equal proportions,
[0099] 1. TATGTATCCATATGACGTCCCAGAC (SEQ ID NO: 7)
[0100] 2. TATGCCGGCGGCAGGAAGAAGCGGAGACAGCGACGAAGAG (SEQ ID NO: 8)
[0101] 3. GATCCTCTTCGTCGCTGTCTCCGCTTC (SEQ ID NO: 9)
[0102] 4. TTCCTGCCGCCGGCATAGTCTGGGACGTCATATGGATACA (SEQ ID NO: 10);
[0103] Denature at 95°C for 10 minutes, then slowly cool down to room temperature for 6 hours, and finally form a double-stranded linker with two restriction enzyme site...
Embodiment 2
[0117] Example 2 Expression of recombinant polypeptide TAP21 in Escherichia coli
[0118] The expression vector pET28a-TAT-TAP21 was introduced into Escherichia coli BL-21(DE3), and the transformant was cultured in a volume of 3 L to express the polypeptide TAP21 and obtain endosomes. The obtained endosomes were dissolved in 50 ml of lysis buffer (8M urea, 20mM NaH 2 PO 4 , 500mM NaCl), centrifuged to obtain the supernatant. Then, the His-tagged polypeptide TAP21 was purified by Ni column (Qiagen) and PD10 column (GE) chromatography (see Figure 4 ). Coomassie blue staining showed that the molecular weight of the polypeptide was about 9kDa, and the purity was about 95% (see Figure 5 ).
Embodiment 3
[0119] Example 3 Inhibition of tumor cell growth by polypeptide TAP21
[0120] The biological activity of the polypeptide TAP21 was detected in vitro. MTT (USB) detection method was used to detect cell proliferation, first 3X10 3 Liver tumor cells BEL7404 and HepG2 (ATCC) were inoculated into 96-well culture plates, and after culturing for one day, different concentrations were added (specific concentrations such as Image 6 A and 6B) TAP21 polypeptide and inactivation mutant TAP21 (TAPm, SEQ ID NO: 17), and PBS was used as a control (control). One day later, add MTT to the well to be tested, select a wavelength of 490nm to detect the absorbance value, and record the result. Draw the cell growth curve with the concentration as the abscissa and the absorbance as the ordinate, and the results are as follows Image 6 A and 6B are shown. In another experiment, the culture medium containing 20 μM polypeptides (TAP21 and TAPm) was changed every day, and PBS was used as a control...
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