Recombinant akabane virus capsid protein, its preparation method and uses
A nucleocapsid protein, Akabane virus technology, applied in the field of genetic engineering, can solve the problems of high technical equipment requirements, maternal antibody interference, complicated operation and other problems, and achieves the effects of easy purification and preparation, guaranteed specificity and low cost
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Embodiment 1
[0040] [Example 1] The construction of recombinant AKAV nucleocapsid protein gene prokaryotic expression vector
[0041] Use RNeasy(R) Mini Kit RNA extraction kit to extract AKAV total RNA, synthesize primers from Saibaisheng Company, amplify the S segment gene, the expected amplification length is 840bp, primer sequence: p1: 5-TAA CTA CGC ATT GCA ATG GC- 3 (SEQ ID NO: 3); p2: 5-AGT AGT GTGCTC CAC-3 (SEQ ID NO: 4). Amplification conditions: 42°C for 1h, 94°C for 5min and then cycle, the cycle parameters are 94°C for 60s, 55°C for 30s, 72°C for 60s, after 30 cycles, 72°C for 10min, 10g / L agarose gel electrophoresis identification PCR product.
[0042] The product purified by PCR was connected to pMD18-T Vector, transformed into E.coli JM109, coated with LB solid medium containing ampicillin (100ug / ml), X-gale and IPTG, picked white single colony after culturing overnight at 37°C, Inoculate into liquid LB containing ampicillin (100ug / ml), incubate on a shaker at 37°C for 8 hou...
Embodiment 2
[0044] [Example 2] Induction condition optimization test
[0045] Transform the recombinant plasmid PET-N into E.coli BL21(DE3) expression host bacteria, pick a single colony from the LB plate and insert it into 3ml LB medium (all medium contains kanamycin), culture overnight at 37°C, and then Insert 100ml of 2×YT medium at 37°C with 150 and culture at 37°C, do the optimization test of induction conditions, use uninduced bacteria as control, do different induction time, different D 600nm The value of the initial induction, different IPTG amount induction test, and finally determine the best induction conditions.
[0046] It can be seen from Figure 6 that different induction times and different D 600nm The value has a significant effect on the expression rate of the fusion protein, but the induction by different IPTG amounts is not obvious. OK D 600nm When the value is 0.5-0.6, IPTG is used to induce the expression of the recombinant nucleocapsid protein, and the IPTG is 0.6...
Embodiment 3
[0047] [Example 3] Identification, purification and expression measurement of recombinant AKAV nucleocapsid protein
[0048] After the engineered bacteria were induced, they were ultrasonically lysed and centrifuged at 12,000×g for 15 minutes. The expressed proteins were detected in the supernatant. Compared with the control, there was a bright band at 27ku, which was consistent with the expected size. ProBond TM After purification by Purification System, only this band remains, which shows that it is indeed an induced fusion protein. Bovine anti-AKAV positive serum was used as the primary antibody, and horseradish peroxidase-labeled rabbit anti-bovine IgG was used as the secondary antibody. After DAB color development, the results showed that there were obvious bands at the corresponding positions, while the control did not, which indicated that the expressed The fusion protein of has immunological activity (Fig. 3, Fig. 4).
[0049] The recombinant fusion protein of the pr...
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