Preparation method of human nerve growth factor transgenic pig

Abstract

The invention discloses a preparation method of a human nerve growth factor transgenic pig. The method comprises the following steps: a, constructing a CRISPR / Cas9 system expression vector of a targeted pNGF gene exon, b, constructing a homologous recombinant vector for providing an hNGF gene, c, co-transfecting porcine ear fibroblasts with the CRISPR / Cas9 system expression vector and the homologous recombinant vector of the hNGF gene, and d, screening the transfected porcine ear fibroblasts to obtain a positive cell line, and cloning somatic cells to obtain the transgenic pig with the human nerve growth factor. According to the invention, by a fixed-point gene recombination mode, the hNGF gene is insert into a pig pNGF gene locus, therefore, the obtained transgenic pig with the human nerve growth factor can express the human nerve growth factor without expressing the pig nerve growth factor, so that the current situation that when the human NGF is collected in pig saliva, the human NGF is mixed in the pig saliva is avoided, the problem of immunogenicity when the pig is used for drug therapy is also avoided, and the transgenic pig is safer and more effective.

Description

technical field [0001] The invention relates to an animal obtained by a transgenic method and a preparation method thereof, in particular to a preparation method of a transgenic pig transformed with human nerve growth factor. Background technique [0002] Nerve growth factor (NGF) is a neurotrophic protein that maintains the survival of neurons, promotes their growth and differentiation, and plays an important role in the development of animals and various physiological processes of adult animals. In addition, NGF plays an important auxiliary role in the growth and activity maintenance of non-nerve cells such as immune cells, fibroblasts, pancreatic cells, and cardiomyocytes, as well as in the repair of tissue damage. Therefore, NGF has good clinical or potential therapeutic effects on many neurological and non-neurological diseases and injuries. [0003] NGF drugs currently on the market are mainly extracted from the submandibular gland of mice, that is, mouse NGF (mouse N...

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Problems solved by technology

[0004] Various methods have been used to obtain hNGF, such as in vitro cultured animal cells, transgenic Escherichia coli and yeast, etc., but due to low production efficiency or the inability to obtain correct post-translational modifications of the protein, these methods have not been implemented in production. application level

Another study used a transgenic animal mammary gland bioreactor to prepare hNGF, which improved the production of hNGF, but there are still defects: mammary gland bioreactor is subject to animal sex (female) and physiological cycle (postpartum lactation period); Contains a large number of protein types and quantities, which is not conducive to the subsequent purification and extraction of hNGF

However, since the endogenous NGF gene of pigs has not been knocked out, pig NGF (pig NGF, pNGF) is still contained in pig saliva, and the physical and chemical properties of pNGF and hNGF are similar, so it is difficult to separate the two

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