Pronuclei fusion expression vector and application thereof
A technology of fusion expression and expression vector, applied in the field of genetic engineering, to achieve the effect of good solubility and easy purification
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Embodiment 1
[0042] Embodiment 1, the construction of fusion expression vector pPDI
[0043] 1. PCR amplification of PDI gene
[0044] PDI is obtained from the Conus snail cDNA library, therefore, the PDI gene was amplified using the Conus snail cDNA as a template by PCR method. According to the analysis of RACE results by computer software, a pair of primers were designed and synthesized:
[0045] The upstream primer is 5’–ATGGCTAGCATGAAGTTTTCATCTTGTT-3’,
[0046] The downstream primer is 5'-GATCCCAGTTCATCTCTTGGCAGATTCTCA-3'.
[0047] For the convenience of cloning, NheI and BamHI restriction enzyme recognition sequences were added to the upstream and downstream primers respectively.
[0048] The PCR reaction was carried out according to the Takara system method.
[0049] 2. Construction of transfer vector pGEM-PDI containing PDI gene
[0050] The PDI gene amplified by PCR was detected and recovered by agarose gel electrophoresis, and then double-cut by Nhe I and BamH I and inserted ...
Embodiment 2
[0057] Example 2. Application of Fusion Expression Vector pPDI
[0058] The conus snail PPI gene and lt14a gene were inserted into the SacI and HindIII restriction restriction windows to construct fusion expression vectors pPDI-PPI and pPDI-lt14a.
[0059] Transform pPDI-PPI, pPDI-lt14a into E. coli BL21C DE3). Inoculate a single colony in 50ml kanamycin-resistant LB medium, culture overnight at 37°C, 250rpm, inoculate 20mL of the overnight culture into 2L ammonium-resistant LB medium, cultivate at 37°C, 250rpm until OD600=0.6 , adding 100mM IPTG and 20% glucose to a final concentration of 1mM and 0.2%, respectively, 18°C, 250rpm induction culture for 14h, and centrifuged to harvest the cells, sonicate the cells, centrifuge to collect the supernatant, and use Ni 2+ Chelating Sepharose affinity chromatography purification. For the expression and purification of pPDI-lt14a fusion protein, see Figure 5 .
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