A method for heat treatment to remove non-VLPs proteins in pcv2 VLPs
A protein and sample technology, which is applied in the field of heat treatment to remove non-VLPs proteins in PCV2VLPs, can solve the problem of small time-consuming and processing volume, and achieve the effect of convenient heat treatment, low cost and obvious effect
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Embodiment 1
[0029] Example 1: Preparation of clear E.coli BL21(DE3) / pET28a-cap bacteria
[0030] Step 1: artificially synthesize the Cap gene, its nucleotide sequence is shown in SEQ ID NO.1, with a Nco I restriction site at the 5' end and a Hind III restriction site at the 3' end.
[0031] Step 2: Use the pET-28α (preserved in our laboratory) vector as the backbone, use Nco I and Hind III to digest and recover the backbone fragment, use Nco I and Hind III to digest and recover the Cap-TFlg fragment, and then use ligase to Ligated overnight at 16°C, and the ligated product was transformed into E. coli From DH5α Escherichia coli competent cells, the colonies were finally picked and dissolved in 10 µL medium for colony PCR identification, and the positive recombinants were extracted into plasmids for enzyme digestion identification and sequencing, and finally the transfer vector pET28a-cap was obtained.
[0032] Among them, PCR identification uses pET-28α vector primers, and its PCR system ...
Embodiment 2
[0049] Example 2: clear E. coli BL21(DE3) / pET28a- cap shake flask expression
[0050] The specific steps are: inoculate 20 μL of clear E.coli BL21(DE3) / pET28a-cap glycerol bacteria into a test tube containing 5 mL of LB liquid medium containing a final concentration of 50 μg / mL Kana, and culture overnight at 37°C on a shaker at 200 rpm ;Transfer 4 mL of the overnight cultured clear E.coli BL21(DE3) / pET28a-cap bacterial solution to a 500 mL conical tube containing 200 mL of LB liquid medium containing a final concentration of 50 μg / mL Kana according to the inoculum size of 2%. flask, 37°C, 200 rpm shaker culture; when OD600=0.6, add 0.4 mM IPTG, 37°C, 200 rpm shaker culture, induce expression for 3 h; after induction, centrifuge at 8000 rpm for 10 min at 4°C to collect bacteria.
Embodiment 3
[0051] Embodiment 3: Preparation of PCV2 VLPs crude sample
[0052] The specific steps are: resuspend bacteria with 1 g of wet bacteria / 10 mL of PBS, ultrasonically crush, crushing power is 300 W, work for 3 s and rest for 5 s, and crush for 10 min in total; centrifuge at 12,000 rpm for 20 min at 4°C to break up the bacteria After supernatant and precipitation, the bacterial supernatant was precipitated with 60% saturated ammonium sulfate for 4 h, centrifuged at 12,000 rpm for 10 min at 4°C to collect the precipitate, and then resuspended with an equal volume of PBS to obtain the crude PCV2 VLPs sample.
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