New preparation of apolipoprotein A-IV
A technology of apolipoprotein and A-IV, which is applied in the field of high-density fermentation to produce recombinant human apolipoprotein A-IV, and achieves the effects of stable expression system, stable strain and simple medium components
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Embodiment 1
[0051] Example 1: Cloning and amplification of human apolipoprotein A-IV gene (hApoA-IV)
[0052] (1) Trizol method extracts total RNA from human small intestine tissue
[0053] Cut the freshly separated human small intestine tissue into 100mg size, and immediately put it in liquid nitrogen for quick freezing. Extract total RNA from tissues as follows. Take out 300 mg of frozen tissue and place it in a mortar filled with liquid nitrogen, and crush the tissue. Move the crushed tissue into a 50ml centrifuge tube, add about 5ml Trizol, and homogenize at high speed with a homogenizer at room temperature for 15-30 seconds. Add 1.0ml chloroform (200μl / ml Trizol), shake well, and place it at room temperature for 5 minutes. After centrifugation (12000r / min) at 4°C for 15 minutes, the upper aqueous phase was transferred to another centrifuge tube. Add an equal volume of isopropanol, shake well, precipitate at room temperature for 10 minutes, centrifuge at 4°C (12000r / min) for 15 minutes...
Embodiment 2
[0089] Example 2: Construction of hApoA-IV Pichia pastoris secretory expression vector pPICZa / hApoA-IV and host cell transformation
[0090] Take 50 ng of the plasmid carrying the hApoA-IV gene identified by the above sequencing, and establish a PCR reaction system in a 0.2ml EP tube according to the above ratio. Using the above PCR reaction system and conditions, the upstream primer 5'-TAACTCGAGAAGAGAGAGGTCAGTGCTGA-3' (SEQ ID NO: 3) was used to introduce the partial sequence of the yeast a factor signal peptide and the XhoI site, and the downstream primer: 5'-TATTCTAGATCAGCTCTCCAAAGGGGCCA-3 '(SEQ ID NO: 4) introduced Xba I site. After PCR introduces the above sequence and restriction site, the target fragment is recovered. After digestion with the corresponding enzymes, connect the plasmid pPICZa which was digested with the same enzymes to construct the Pichia expression vector pPICZa / hApoA-IV, and then transform into Escherichia coli, extract the plasmids and conduct enzyme di...
Embodiment 3
[0094] Example 3: Expression of ApoA-IV polypeptide
[0095] Take the above-mentioned positive clones and inoculate them in 10ml BMGY (pH 6.0) medium, culture with shaking at 30°C for 24 hours, to OD 600 Collect cells when it reaches 2.0 to 6.0. Resuspend the cell pellet with an equal volume (10ml) of BMMY (pH 6.0), culture with shaking at 30°C to induce expression. During the induction process, methanol was added every 24 hours to a final concentration of 0.5%, and sterilized ultrapure water was added to keep the total volume of the fermentation broth unchanged. At 0, 24, 48, 72, 96, 120, 144, 168 hours of culture, 0.5ml of fermentation broth was taken, and the supernatant was centrifuged for protein analysis (SDS-PAGE, N-terminal amino acid sequence analysis, etc.) ).
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