TIPE-2 and NRDP1 recombinant compound and preparation method and application thereof
A 1.TIPE-2, complex technology, applied in botanical equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of difficult evaluation and analysis of experimental results, time-consuming, and many influencing factors.
Pending Publication Date: 2020-10-30
AFFILIATED YONGCHUAN HOSPITAL OF CHONGQING MEDICAL UNIV
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Problems solved by technology
[0005] There are two main methods of gene combination therapy: one is to simultaneously transfect target cells with a variety of recombinant expression vectors carrying different genes. The construction of recombinant expression vectors is cumbersome, time-consuming, and has many influencing factors. In addition, due to the randomness of the transfection process, the transfected cells have the problem of uneven gene copies, which is not conducive to the expression of the target gene and subsequent treatment. It makes the experimental results difficult to evaluate and analyze; the above shortcomings limit the further application of this method
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[0045] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The experimental method that does not indicate specific conditions in the preferred embodiment is usually according to conventional conditions, such as described in the Molecular Cloning Experiment Guide (Third Edition, J. Sambrook et al., translated by Huang Peitang, etc., Science Press, 2002) conditions, or as recommended by the manufacturer.
[0046] 1. Preparation of recombinant complex Ad-TIPE-2 / NRDP1
[0047] 1. Cloning of TIPE-2 RNAi
[0048] According to the instructions of the Trizol kit, the total RNA of human liver tissue was extracted, and the total cDNA was obtained by reverse transcription. Then, using the total cDNA as a template, F1: 5'-ttttcgcccgcacagagcggaatctgcaga-3' (SEQ ID No.1) and R1: 5'- gcacgaggagggttacgaattcaagcgcgtcacg-3' (SEQ ID No.2) is the upstream and downstream primers for PCR amplification of TIPE-2 full-...
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The invention discloses a TIPE-2 and NRDP1 recombinant compound and a preparation method and application thereof. The recombinant compound is composed of a polypeptide compound and a double-gene expression cassette. The polypeptide compound is composed of a cell-penetrating peptide Transsortan9 (TP9) LKALLGKIN and a positive charge polypeptide KKKKKKKKK, the double-gene expression cassette comprises a TIPE-2 RNAi and NRDP1 double-gene expression cassette, and the expression cassette sequentially comprises a CMV promoter, a TIPE-2 gene, a terminator, an internal ribosome entry site IRES, an NRDP1 gene and a terminator from upstream to downstream; the preparation method of the recombinant compound is simple, the recombinant compound can inhibit macrophage activation, effectively inhibit inflammatory response and encephaledema of cerebral hemorrhage and inhibit nerve energy supply damage when used for in-vivo transfusion of peripheral tissues of hematoma of cerebral hemorrhage, and provides an ideal strategy for treatment of cerebral hemorrhage.
Description
technical field [0001] The present invention relates to a recombinant complex, in particular to a recombinant complex of TIPE-2 and NRDP1, and also relates to a preparation method and application of the recombinant complex. Background technique [0002] Intracerebral hemorrhage (ICH) is a common clinical cerebrovascular disease, lack of effective treatment, high mortality and disability rates. The secondary neurological injury a few days after cerebral hemorrhage is a critical period of exacerbation. The mechanism of secondary nerve injury is complex and may involve the formation of perihematomal cerebral edema, abnormal metabolism around hematoma, and changes in regional cerebral blood flow (rCBF) around hematoma. Related studies have shown that the inflammatory response mediated by macrophages plays an important role in the secondary nerve injury of cerebral hemorrhage. Macrophages are highly heterogeneous, and changes in the local microenvironment can lead to different ...
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IPC IPC(8): A61K48/00A61K38/08A61K47/42A61K39/00A61P7/04A61P25/00A61P29/00A61P7/10C12N15/861C12N15/66C12N15/113C12N15/52C12N7/01C12R1/93
CPCA61K48/005A61K48/0008A61K38/08A61K47/42A61K39/00A61P7/04A61P25/00A61P29/00A61P7/10C12N15/86C12N15/66C12N15/113C12N9/93C12N7/00C12Y603/02019C12N2710/10043C12N2710/10052C12N2710/10021C12N2800/107C12N2840/203C12N2310/14A61K2300/00
Inventor 杨曌
Owner AFFILIATED YONGCHUAN HOSPITAL OF CHONGQING MEDICAL UNIV