TPA gene thread and its preparation method
A suture and gene technology, applied in the medical field, can solve the problems of structure and preparation method description, and achieve the effects of eliminating or avoiding the formation of blood clots, enhancing fibrinolytic activity, and prolonging survival time
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Embodiment 1
[0022] Example 1 Preparation of tPA gene suture
[0023] 1. Main reagents
[0024] Plasmid pSecTag2B was purchased from Invitrogen Biological Company of the United States, and competent cells E.ColiJM109 were purchased from Promega Company. Restriction enzymes HindIII, KpnI, BamHI and XhoI were purchased from Bao Biological Engineering Co., Ltd., T 4 - DNA ligase was purchased from New England Biolabs, and QIAquick Gel Extraction Kit and QIAGEN PCR Product Purification Kit were purchased from QIAGEN.
[0025] 2. Method
[0026] 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 plasmid pOTB6 , pOTB7 and pCMV-SPORT6.
[0027] Three pairs of primers t-PA-1F (SEQ ID NO.5) and t-PA-1R (SEQ ID NO.6), t-PA-2F (SEQ ID NO.7) and t-PA-2R (SEQ ID NO. NO.8), t-PA-3F (SEQ ID NO.9) and t-PA-3R (SEQ ID NO.10) amplify plasmids pOTB6, pOTB7 and pCMV-...
Embodiment 2
[0036] Example 2 Effect of tPA gene suture in preventing thrombosis after cardiac surgery
[0037] 1. Materials
[0038] Animals: 36 healthy pigs, weighing 40-50KG, female, 8 months old. Among them, 18 were used for blood supply, and the other 18 were randomly divided into 9 in the control group and 9 in the experimental group.
[0039] 2. Method
[0040] 2.1 Pig tricuspid valve replacement and transgene therapy
[0041] The control group underwent simple tricuspid valve replacement under conventional extracorporeal circulation; the experimental group sutured the prepared tPA gene suture serpentinely or circularly between the myocardial wall of the right ventricular outflow tract after tricuspid valve replacement, and the length of the gene suture About 10cm, at the same time, perform ultrasonic treatment on the part of the gene suture, frequency: 1kHz, intensity: 0.5w / cm 2 , time: 30min. At 1 week, 2 weeks, 4 weeks, 8 weeks and 12 weeks after operation, venous blood was ...
PUM
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