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Recombinant human lactoferrin (rhLF) silkworm chrysalis powder as well as preparation method and application thereof

A technology of silkworm chrysalis powder and protein, which is applied in the field of biomedicine, can solve the problems of restricting drug efficacy, inconvenient application of dosage forms, and low bioavailability, and achieve good medicinal value, high application value, and good biological activity.

Inactive Publication Date: 2015-04-29
TIANJIN YAOYU BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Commonly used drug preparations for the treatment of UC include liquid enemas, foams and suppositories, but the inconvenient application of the dosage forms restricts the efficacy of the drugs.
The oral preparations of traditional Chinese medicine are mostly decoctions, and most of them are crude preparations, with large dosage, low bioavailability and slow drug effect
Sulfasalazine Tablets (SASP) is currently the preferred oral drug for the treatment of mild to moderate UC by Western medicine. After oral administration, it is decomposed into 5-aminosalicylic acid and sulfadiazine by bacteria in the intestinal tract. The drug reaches the colon; the latter has no therapeutic effect and produces a large number of adverse reactions after absorption

Method used

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  • Recombinant human lactoferrin (rhLF) silkworm chrysalis powder as well as preparation method and application thereof
  • Recombinant human lactoferrin (rhLF) silkworm chrysalis powder as well as preparation method and application thereof
  • Recombinant human lactoferrin (rhLF) silkworm chrysalis powder as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: Obtaining of hLF gene (its nucleotide sequence is shown in SEQ ID NO:1)

[0043]Design a pair of primers according to the gene sequence of hLF (gene accession number: AY875691.1) provided on NCBI:

[0044] hLF F: 5'-GCGCTCGAGATGAAACTTGTCTTCC-3'

[0045] hLF R: 5'-CGCGGTACCTTACTTCCTGAGGAATTC-3'

[0046] Recombinant Functional Human Lactoferrin Expressed in Baculovirus System(J). Acta Biochimica et Biophysica Sinica, 2006, 38 (3): 201– 206) as a template, with hLF F and hLF R as upstream and downstream primers, PCR amplification is carried out, and the specific reaction system and reaction procedure are as follows:

[0047] PCR reaction system:

[0048] 10×Taq buffer 5 μL

[0049] 10 mM dNTPs 1 μL

[0050] hLFF 2.5 μL

[0051] hLFR 2.5 μL

[0052] Template 1 μL

[0053] Taq enzyme 1 μL

[0054] wxya 2 O 37 μL

[0055] Reaction procedure:

[0056]

[0057] After the reaction was completed, the amplified fragment was identified by electrophoresi...

Embodiment 2

[0058] Example 2: Construction of recombinant transfer plasmid pFastBac1-hLF

[0059] The pFastBac 1 vector (purchased from Invitrogen) was used xho I and KpnI (purchased from Fermentas Company) After double enzyme digestion, the large fragment was recovered and ligated with the purified hLF PCR product to construct the recombinant transfer plasmid pFstBac1-hLF. The correct gene sequence was identified by restriction analysis and bidirectional sequencing, indicating that the recombinant transfer plasmid was successfully constructed. figure 2 It is the electrophoresis figure of the double enzyme digestion and PCR identification of the recombinant transfer plasmid pFastBac1-hLF.

Embodiment 3

[0060] Embodiment 3: Obtaining of Bombyx mori recombinant baculovirus Bm-rhLF

[0061] The recombinant transfer plasmid pFastBac1-hLF, which was identified as a successful recombination, was transformed into Escherichia coli DH10Bac competent cells containing the baculovirus shuttle vector Bacmid (purchased from Invitrogen Company), in the presence of kanamycin, gentamicin, tetracycline, X-gal and IPTG was cultured on an LB culture plate (purchased from Shanghai Sangon Biotechnology Co., Ltd., operated according to the instructions), and homologous recombination was carried out by transposition (the hLF sequence on pFastBac 1-hLF was inserted into the multi-cloning site of Bacmid by homologous transposition After 48 hours of culture in the dark, the white spot was picked, and the white spot was continued to be shaken in the LB culture medium containing tetracycline, kanamycin, gentamycin, X-gal and IPTG for 48 hours, and then treated with isocyanide The recombinant baculovirus...

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Abstract

The invention relates to recombinant human lactoferrin (rhLF) silkworm chrysalis powder as well as a preparation method and application thereof and belongs to the technical field of biological medicines. A recombinant virus is constructed mainly by adopting a silkworm chrysalis baculovirus expression system, the rhLF is expressed with a silkworm chrysalis serving as a bioreactor, the silkworm chrysalis diseased after being inoculated with the recombinant virus is prepared into freeze-dried powder, the activity of the freeze-dried powder is detected by performing antibacterial experiments in vitro, and the freeze-dried powder is poured into the stomach of a mouse to treat the dextran sulphate sodium (DSS) induced ulcerative colitis of the mouse. The analysis on the disease activity index (DAI) of the mouse and the detection on the content of myeloperoxidase (MPO) in the colonic tissue of the mouse show that the rhLF with antibacterial activity is expressed successfully in the silkworm chrysalis, and the rhLF silkworm chrysalis powder has a certain effect of treating the DSS induced ulcerative colitis of the mouse. The preparation method provided by the invention is simple, is low in cost and is used for preparing a novel oral protein medicine.

Description

technical field [0001] The invention relates to recombinant human lactoferrin silkworm chrysalis powder and its preparation method and application, belonging to the technical field of biomedicine. Background technique [0002] Lactoferrin (Lactoferrin, LF for short), is a member of the transferrin genus, mainly exists in mammalian whey, also known as lactoferrin. Lactoferrin is widely distributed. In addition to the abundant lactoferrin in milk, it is also detected in some secretions, such as tears, semen, and bile. [0003] Human lactoferrin (Human Lactoferrin, hLF), a secreted iron-binding glycoprotein, is folded into two lobe structures with similar structures by a polypeptide chain, and each structure contains an iron-binding site. The cDNA of hLF is 2136bp, the first 19 amino acids encoded are signal peptide, and the last 692 amino acids constitute mature human lactoferrin with a molecular weight of about 80kD. hLF has various biological functions such as iron binding...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N15/88C12Q1/18A61K38/40A61P1/04C12R1/19
Inventor 张耀洲赵红玉舒特俊盖其静李亚宾
Owner TIANJIN YAOYU BIOLOGICAL TECH
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