Methods of Expressing Polypeptides

An insect and cell technology, applied in the field of FMRP protein expression and expression of polypeptides, can solve the problems that need to be deepened and purify FMRP protein, and achieve the effect of simple operation, eliminating the interference of human subjective factors, and high sensitivity.

Active Publication Date: 2016-10-19
NAT RES INST FOR FAMILY PLANNING
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Problems solved by technology

[0004] Expression and purification of high-yield and high-purity FMRP protein is an important experimental material for the rapid and high-throughput detection method of FMRP, and it is also the basis for the preparation of FMRP monoclonal antibodies and the development of FXS detection kits. However, there is no method to express , Purification to obtain high-yield, high-purity FMRP protein
[0005] Therefore, the research on the expression method of FMRP protein still needs to be further studied.

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  • Methods of Expressing Polypeptides
  • Methods of Expressing Polypeptides
  • Methods of Expressing Polypeptides

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Embodiment 1

[0061] 1. Materials

[0062] Cell line: Sf9 insect cells, purchased from the Basic Medical Cell Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

[0063] Eukaryotic donor plasmid: pFastBac1 was purchased from Invitrogen.

[0064] Strains: Escherichia coli TOP10 competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd., and Escherichia coli DH10Bac competent cells were purchased from Invitrogen.

[0065] Primer: Synthesized by Invitrogen, the sequence (5' to 3') is as follows:

[0066] Middle-Forward: TTGACATGCACTTTCGGA (SEQ ID NO: 3),

[0067] pUC / M13-Forward: CCCAGTCACGACGTTGTAAAACG (SEQ ID NO: 4),

[0068] pUC / M13-Reverse: AGCGGATAACAATTTCACACAGG (SEQ ID NO:5),

[0069] Forward: CCG GAATTC ATGGAGGAGCTGGTGGTG (SEQ ID NO: 6),

[0070] ISO10-Reverse: CGC GTC GAC TTA GTGATGGTGATGGTGATG ATGACCAATCACTGA

[0071] (SEQ ID NO: 7),

[0072] Among them, the 5' end of the primer Forward introduces the restriction endonuc...

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Abstract

The invention discloses a method for expressing polypeptide, wherein the polypeptide has an amino acid sequence shown as SEQ ID NO:1. The method for expressing polypeptide comprises the following steps of: infecting a first insect Sf9 cell by an expression virus seed of recombinant baculovirus, wherein the first insect Sf9 cell expresses polypeptide, and the expression virus seed of recombinant baculovirus comprises nucleic acid molecules which code the polypeptide; and cracking the first insect Sf9 cell and purifying to obtain the polypeptide. According to the method provided by the invention, the polypeptide can be effectively expressed to further obtain FMRP (Fragile X Mental Retardation Protein) with high yield and high purity.

Description

technical field [0001] The present invention relates to the field of biotechnology, specifically, to a method for expressing polypeptides, and further, to a method for expressing FMRP protein. Background technique [0002] Fragile X syndrome (fragile X syndrome, FXS) is a common hereditary mental retardation syndrome, its incidence is second only to Down syndrome, and it is X-linked inheritance. The clinical manifestations are different degrees of mental retardation, long face, big ears, huge testicles after puberty, etc. Due to the large difference in clinical manifestations, it is difficult to make a correct diagnosis based on clinical phenotype alone. The incidence of FXS is 1 / 4000 male and 1 / 6000 female. FXS patients account for 2.6% to 8.7% of mentally handicapped people, which brings heavy psychological and economic burdens to families and society, and there is no effective treatment at present. Therefore, FXS screening is very important at home and abroad. Early det...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/866
Inventor 郭永邹检平李芳芳张瑶楠
Owner NAT RES INST FOR FAMILY PLANNING
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