Type-S non-virus carrier and pharmaceutical composition containing the same
A technology of non-viral vectors and compositions, which can be applied in antiviral agents, hybrid peptides, pharmaceutical formulations, etc., and can solve the problems of being unable to be promoted in clinical practice, unable to be truly promoted, and poor targeting
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Embodiment 1
[0042] The present invention is further described by the following examples. Example 1 Cloning of PEIIImut and Construction of Expressed Recombinants 1. PEIII mut Amplification and cloning of
[0043] Using the plasmid pZM3 containing the Pseudomonas aeruginosa toxin PE40 as a template, the coding sequence of the III domain of the Pseudomonas aeruginosa toxin was amplified by PCR with the following primers. The recognition sequences of EcoRV and EcoRI have been introduced in the primer sequences used, and the codons of the 5 amino acids mentioned above at the 3′ end of the PEIII coding sequence have been mutated into 4 amino acid codons. The primer sequences are: Upstream primer (33bp): 5'-tc gatatcaccatg ctcggcgacggcggcgacg-3′
[0044] EcoRV Kozak downstream primer (40bp): 5′-g gaattc ttacagttcgtccttcggcggtttgccggggctg-3′
[0045] The EcoRIPCR reaction conditions were as follows: denaturation at 94°C for 5 minutes followed by cycling: de...
Embodiment 2
[0046] The target DNA fragment recovered above was connected with the DNA fragment of the mammalian expression vector pcDNA3.1(-) / Myc-HisA which was also treated with EcoRV and EcoRI, and transformed into Escherichia coli DH5α. Positive clones were screened out, plasmids were prepared from them, and the obtained plasmids were identified by digestion with EcoRV and EcoRI. Thus, a positive recombinant plasmid containing about 650bp insert fragment was identified and named as pcDNA3.1-PEIII mut , its construction process is as follows figure 2 shown. The resulting PEIII was sequenced mut The sequences of the fragments coincided with the designed sequences. PEIII mut The size is 642bp, and its sequence is shown in SEQ ID NO.:7. Example 2 Recombinant plasmid pYL-PEIII mut Construction 1, pBS-PEIII mut build
[0047] Use EcoRV and HindIII (Liuhetong Company, Dalian, China) to contain PEIII mut Vector pcDNA3.1-PEIII of the DNA fragment of the toxin gene mut (See Example 1)...
Embodiment 3
[0048] pYL158 is an expression plasmid containing the fragment of HIV-1 5'-LTR-158 to +80 as a promoter and luciferase reporter gene. Use PstI and ClaI (Liuhetong Company, Dalian, China) to combine PEIII mut DNA fragment from pBS-PEIII mut It was digested with the middle enzyme, and connected with the pYL158 vector fragment treated by the same enzyme digestion. Transform Escherichia coli DH5α by taking part of the ligation product, screen out positive clones, and prepare a small amount of plasmids for the positive clones, carry out enzyme digestion and identification of the plasmids, and confirm that there is PEIII mut The positive recombinant plasmid with correct fragment insertion is named pYL-PEIII mut , its construction process is as follows Figure 4 shown. Example 3 Artificial synthesis of mutant gene encoding CD4V1 region
[0049] In order to obtain high expression in Escherichia coli, according to the codon preference principle in bacteria, all the codons not pref...
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