A kind of oral porcine growth hormone fusion protein and its application
A pig feed and feed additive technology, applied in the field of genetic engineering, to increase productivity, improve daily weight gain and feed-to-meat ratio indicators
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Embodiment 1
[0045] Embodiment 1 Utilizes Escherichia coli to prepare oral porcine growth hormone fusion protein
[0046] According to the Tat PTD sequence and the porcine growth hormone mature peptide amino acid sequence, the amino acid sequence of the porcine growth hormone fusion protein (SEQ ID No.1) is designed. The base sequence SEQ ID No.2 was designed according to the sequence of SEQ ID No.1 and considering the codon preference of Escherichia coli. NdeI and EcoRI restriction sites were designed before and after SEQ ID No.2, and Sangon Bioengineering (Shanghai) Co., Ltd. was entrusted to carry out the whole gene synthesis.
[0047] The synthetic whole gene sequence was digested with NdeI and EcoRI and the digested fragment was recovered, and then ligated with the pET22b plasmid vector fragment which was also digested and recovered. The ligation product was transformed into Escherichia coli DH5α competent cells by the calcium chloride method, and the positive transformants were scre...
Embodiment 2
[0052] Example 2 Oral porcine growth hormone fusion protein oral penetration activity test
[0053] The experimental animals were SD rats (purchased from the Experimental Animal Center of Anhui Medical University), 3 males, weighing about 200 grams. Animals were fasted without food and water for 12 hours before the start of the experiment. Take the orally administered porcine growth hormone fusion protein stock solution prepared in Example 1, dilute it to 1 mg / ml with PBS, and administer 1 mg of porcine growth hormone fusion protein by intragastric administration to each animal. Before the test (0 hour) and 0.5, 1, 2, 3, and 4 hours after gavage, 500 microliters of blood were collected from the orbit, rested in ice bath for 1 hour, centrifuged at 4000 rpm to separate the serum, and frozen at minus 70°C.
[0054] According to the instructions of Cusabio kit (CSB-E06813p), the content of porcine growth hormone fusion protein in rat serum was determined. Experimental results su...
Embodiment 3
[0055] Example 3 Utilize Pichia pastoris to prepare oral porcine growth hormone fusion protein
[0056] According to the amino acid sequence of SEQ ID No.1 and the codon preference of Pichia pastoris, the base sequence shown in SEQ ID No.3 was designed, EcoRI and XhoI restriction sites were added before and after the sequence of SEQ ID No.3, and Entrust Sangon Bioengineering (Shanghai) Co., Ltd. to carry out the whole gene synthesis.
[0057] The synthetic whole gene sequence was digested with EcoRI and XhoI and the digested fragments were recovered, and then ligated with the pGAPZ plasmid vector fragments that were also digested and recovered. The ligation product was transformed into Escherichia coli DH5α competent cells by the calcium chloride method, and the positive transformants were screened with an LB resistance plate containing 100 μg / mL ampicillin, and identified by sequencing. A large number of positive transformant plasmids pGAPZ-Tat-pGH with correct sequencing we...
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