Recomposed tPA gene-chitosan nanoparticle complex and preparation method thereof
A technology of chitosan nanoparticles and complexes, applied in gene therapy, drug combination, pharmaceutical formula, etc., can solve the problems of life-long medication, high cost of treatment, high price, etc., to eliminate or avoid the formation of blood clots , improve bioavailability, reduce the effect of thrombus formation
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[0031] The preparation method of recombinant tPA gene-chitosan nanoparticle complex of the present invention mainly comprises the following steps:
[0032] 1. Obtain the recombinant tPA gene plasmid pSecTag2B-tPA, which will be described in detail in Example 1 below;
[0033] 2. Using the above-mentioned recombinant tPA gene plasmid and chitosan to prepare the recombinant tPA gene-chitosan nanoparticle complex, the following example 2 will describe in detail.
Embodiment 1
[0034] Example 1: Preparation of recombinant tPA gene plasmid pSecTag2B-tPA
[0035] 1. Main reagents
[0036] A. Plasmid pSecTag2B, purchased from Invitrogen Biological Company, USA,
[0037] B. Competent cells E.Coli JM109, purchased from Promega,
[0038] C. Restriction enzymes HindIII, KpnI, BamHI and XhoI were purchased from Bao Biological Engineering Co., Ltd.,
[0039] D, T4-DNA ligase, purchased from New England Biolabs company,
[0040] E, QIAquick Gel Extraction Kit, QIAGEN PCR Product Purification Kit, purchased from QIAGEN Company.
[0041] 2. Method
[0042] Three ESTs were artificially synthesized: tPA-1 as shown in SEQ ID NO.2, tPA-2 as shown in SEQ ID NO.3 and tPA-3 as shown in SEQ ID NO.4, respectively connected to cloning vectors to prepare plasmids pOTB6, pOTB7 and pCMV-SPORT6.
[0043] Using three pairs of primers tPA-1F (SEQ ID NO.5) and tPA-1R (SEQ ID NO.6), tPA-2F (SEQ ID NO.7) and tPA-2R (SEQ ID NO.8), tPA -3F (SEQ ID NO.9) and tPA-3R (SEQ ID NO....
Embodiment 2
[0046] Embodiment 2: Preparation method of recombinant tPA gene-chitosan nanoparticle complex
[0047] Utilize the recombinant tPA gene plasmid pSecTag2B-tPA and chitosan that embodiment 1 makes to prepare recombinant tPA gene-chitosan nanoparticle complex, comprising the following steps:
[0048] 1) Preparation of chitosan nanoparticles:
[0049] a) prepare 1.5mg / ml chitosan acetic acid solution, wherein the mass ratio of acetic acid to chitosan is 1.5: 1, first measure 2ml analytical pure glacial acetic acid solution, dissolve it in 38ml triple distilled water, and then weigh 60mg of chitosan powder was dissolved in 40ml of the above acetic acid solution to obtain a 1.5mg / ml chitosan acetic acid solution;
[0050] b) 20ml of polyvinyl alcohol aqueous solution with a concentration of 10mg / ml was slowly dripped into 40ml of chitosan acetic acid solution obtained in step a) under the condition of magnetic stirring, and continued magnetic stirring for 1 hour at normal temperature...
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