Preparation method for chicken interferon-alpha
A technology of chicken interferon and alpha gene, which is applied in the field of preparation of chicken interferon alpha, can solve problems such as lack of biological activity, and achieve the effects of simple expression scheme, favorable absorption and high yield
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Embodiment 1
[0031] Embodiment 1 The acquisition of chicken interferon-alpha gene
[0032] According to the gallus gallus gene sequence reported in the literature (GenBank: U07868.1), the gene expression sequence was optimized, and the optimized sequence is shown in Sequence Table 1, and 15 complementary oligonucleotides were synthesized according to the conventional method of molecular cloning , firstly treated with T4 phage polynucleotide kinase at 37°C for 30min, the phosphorylated oligonucleotide fragments were mixed in equimolarity, denatured at 94°C for 5min, immediately annealed at 65°C for 10min, then added T4 ligase, ligated overnight at 16°C, Obtain target gene template fragments. Take 4 sterile microcentrifuge tubes and add:
[0033]
[0034] The above mixture was shaken gently to mix well, then briefly centrifuged, and then placed in a water bath at 15°C to incubate and connect overnight (12-16h). Take 5 μl of the ligation product and add it to 50 μl of E. coli TOP10 compe...
Embodiment 2
[0035] Example 2 Expression of Chicken Interferon-α Gene
[0036] Design a pair of primers. Primers 1 and 2 are shown in Sequence Listing Sequence 2 and Sequence 3 respectively. The primer at the 5' end of the gene has an Nde I restriction site, and the primer at the 3' end has an EcoR I restriction site. In 0.2ml PCR Prepare a 25μl reaction system in a microcentrifuge tube:
[0037]
[0038]
[0039] Pre-denatured at 94°C for 5 minutes, set 94°C for 1min, 56°C for 1min, 72°C for 2min, a total of 35 cycles, and finally 72°C for 15min. After PCR, 10 μl of the product was taken for agarose gel electrophoresis. The size of the fragment was consistent with the designed fragment size of about 500 bp.
[0040] The pET-32a plasmid was digested with Nde I and Ecor I to recover the large fragment, and ligated with the chicken interferon gene fragment of PCR. The ratio of the gene fragment to the large vector fragment in the 20 μL reaction system was 10:1, and 300 units of T4 DNA...
Embodiment 3
[0043] Example 3 Separation and Purification of Chicken Interferon-α
[0044] 1. Bacteria culture
[0045] Prepare 1 liter of LB medium (tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, pH 7.0), and after the medium is prepared, sterilize it at 121°C for 40 minutes. After the sterilization, add Amp to the ultra-clean bench to make the final concentration of 50 μg / mL when the culture medium is cooled and not hot, and put it in the refrigerator at 4 °C after cooling for later use.
[0046] Under aseptic conditions, a single colony of pET-32a-chIFN-α was picked from the plate and placed in 100ml LB medium (containing Aamp), and then cultured overnight at 37°C on a shaker at 250rpm. The seed culture cultured overnight was transferred to LB medium according to 0.5% inoculation amount, and cultivated at 37°C and 250rpm for 4 hours. L. The culture was continued for 5 hours, and the bacterial cells were collected by centrifugation (5,000 rpm×10 min). The cells were washe...
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