Method for producing transgene protein medicine of mammary gland expression using gland virus as carrier mammary

[0050] Example 1

[0051] Use adenovirus as a vector to introduce goat mammary gland to express and produce human lactoferrin.

[0052] (1) Isolation and cloning of target drug protein gene sequence

[0053] 1. Extraction of total RNA from human breast tissue

[0054] (1) Cut a small piece of frozen breast tissue, quickly weigh it, and then transfer it into the freshly prepared lysis buffer, and add 1 ml of lysis buffer per 50 mg of tissue.

[0055] (2) Homogenize the breast tissue to break the cells, pre-filter and centrifuge once, and collect the filtrate.

[0056] (3) Add an equal volume of chloroform, shake and mix, centrifuge and aspirate the supernatant.

[0057] (4) Add an equal volume of 70% ethanol, mix gently, and centrifuge to obtain total RNA.

[0058] (5) Dissolve the precipitate in Rnase-free water treated with 0.01% DEPC, and perform content determination by conventional methods.

[0059] 2. Synthesis of total cDNA by reverse transcription

[0060] (1) Take 1-2ug of to

[0143] Example 2

[0144] Use adenovirus as a vector to introduce rabbit mammary glands to express and produce human growth hormone. The rabbits used in the experiment were from Changli County, Hebei Province. The amount of vector introduced into the mammary glands of the rabbits was calculated based on the breast capacity of the animals themselves. Among them, the target protein human growth hormone content obtained in step 5 is higher than the expression level of recombinant protein in goat milk, which is 2-8g / L. The other operation methods are the same as those in the steps (1), (2), (3) and (4) of Example 1.

[0145] Example 3

[0146] Adenovirus is used as a vector to introduce porcine mammary gland to express and produce human growth hormone. The pigs used in the experiment were from Changli, Hebei. The amount of vector introduced into the mammary glands of pigs required a small amount of multi-nipple injection. Among them, the target protein hGH expression obtained in step 5 is 0.3g-0.4g / L.