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Cow inhibin alpha subunit mature peptide gene, and recombinant expression protein method

A gene recombination and inhibin technology, applied in genetic engineering, plant gene improvement, recombinant DNA technology, etc., can solve the problems of increasing the difficulty and cost of the test, and the lack of natural activity of the protein, so as to reduce the technical difficulty and cost, and achieve good reaction. Sexuality, beneficial to industrialization

Active Publication Date: 2016-02-03
BEIJING UNIV OF AGRI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, the inclusion body protein is induced in the recombinant inhibin test, and the inclusion body protein needs to be refolded in the subsequent test operation, and the protein after refolding has no natural activity, which greatly increases the difficulty and cost of the test

Method used

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  • Cow inhibin alpha subunit mature peptide gene, and recombinant expression protein method
  • Cow inhibin alpha subunit mature peptide gene, and recombinant expression protein method
  • Cow inhibin alpha subunit mature peptide gene, and recombinant expression protein method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Embodiment 1, bovine InhibinpET32a(+)-matpeptide recombinant vector construction

[0116] 1. Primer design

[0117] Referring to the complete sequence of Bostaurusinhibin, alpha (INHA), NM_174094.4 gene nucleotide matpeptide published on GenBank, a pair of upstream and downstream primers were designed using the primer design software Primer5.0, and 405 nucleotides at the 3′ end were cut out. The sequence of SEQ ID NO: 1 in the sequence listing was obtained.

[0118] Upstream primer P1: 5'-cgggatcccgctccacgcccccactg-3' (26bp) (SEQ ID NO: 3)

[0119] Downstream primer P2: 5'-ccgctcgagaatcccttagatgcaagcaca-3' (30bp) (SEQ ID NO: 4).

[0120] 2. RT-PCR amplification of bovine inhibin matpeptide fragment

[0121] (1) Extraction of bovine ovarian follicle RNA

[0122] ①The ovaries obtained from the slaughterhouse were cut with scissors to cut out the surrounding tissues of the follicles, quick-frozen in liquid nitrogen, and stored at -80°C for later use.

[0123] Soak all...

Embodiment 2

[0178] Embodiment 2: Expression of bovine pET32a(+)-matpeptide-E.coliBL21(DE3) genetic engineering bacteria

[0179] 1. Transformation of recombinant plasmids into host bacteria;

[0180] The recombinant plasmid sequenced correctly in Example 1 was transformed according to the transformation steps, and the LB liquid medium, LB solid medium and AMP storage solution required for transformation were formulated as follows:

[0181] ①LB liquid culture medium

[0182] Tryptone………………………… 1.0g

[0183] YeastExtract……………………0.5g

[0184] NaCl……………………………… 1.0g

[0185] Make up to 100ml with distilled water

[0186] ②LB solid medium

[0187] Tryptone………………………… 1.0g

[0188] Yeast Extract………………… 0.5g

[0189] NaCl……………………………… 1.0g

[0190] Powderedagar……………………1.5g

[0191] Make up to 100ml with distilled water

[0192] ③AMP storage solution

[0193] Weigh 0.5g of Amp and dissolve it in 5ml of deionized water, filter and sterilize through a 0.22um filter, aliquot and store at -20...

Embodiment 3

[0216] Example 3. Application and Immunological Detection of Bovine Inhibin α Subunit Mature Peptide Recombinant Protein of the Present Invention

[0217] In order to obtain the anti-inhibin α subunit rabbit polyclonal antibody with higher titer and increase the ovulation rate of animals, the purified bovine inhibin α subunit recombinant protein of the present invention obtained in Example 2 was used to actively immunize rabbits, blood was collected from the heart and The serum was separated, and the antibody titer was detected by indirect ELISA method to determine whether the corresponding antibody was produced.

[0218] Among them, the rabbit anti-bovine inhibin α subunit polyclonal antibody serum is prepared by the following method:

[0219] The protein obtained in Example 2 was used as an antigen to actively immunize rabbits to prepare polyclonal antiserum.

[0220] 1. Preparation of antigen in Freund's adjuvant by emulsification method

[0221] Boil a glass syringe, a 1...

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Abstract

The invention provides a cow inhibin alpha subunit mature peptide gene extracted from bovine ovaries, and proteins coded by the cow inhibin alpha subunit mature peptide gene. The polynucleotide sequence of the cow inhibin alpha subunit mature peptide gene is represented by SEQ ID NO:1, and amino acid sequence is represented by SEQ ID NO:2. The invention also provides a cow inhibin alpha subunit mature peptide gene recombinant expression vector which contains the cow inhibin alpha subunit mature peptide gene and pET32a(+) vector, and a construction method thereof, and also provides an induction and preparation method of the cow inhibin alpha subunit mature peptide recombinant expression protein.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a bovine inhibin α subunit mature peptide gene, bovine pET32a(+)-matpeptide recombinant vector and pET32a(+)-matpeptide-E.coliBL21(DE3) engineering bacteria The construction of the recombinant protein, the induced expression method and the purification method, as well as the method of preparing polyclonal antibody and immunological detection. Background technique [0002] Superovulation is one of the key technologies for embryo transfer. Currently, the commonly used superovulation drug is mainly FSH. However, because superovulation is a complex physiological process, coupled with the different batch numbers of FSH manufacturers and the differences in individual animals, the production of FSH may be difficult. The super-exhaust effect is not stable. At the same time, due to the short half-life of FSH in the body, frequent injections are required. Compared with supero...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/47C12N15/70C12N1/21
Inventor 倪和民吴迪郭勇邢书涵常迪盛熙晖齐晓龙刘迪
Owner BEIJING UNIV OF AGRI
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