Compositions and methods for treating intracellular diseases
a technology for intracellular diseases and compositions, applied in the direction of antibody medical ingredients, dsdna viruses, peptide sources, etc., can solve the problems of inability to always carry vaccination, lack of adequate sanitation, and serious concern about infectious diseases
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example 1
Isolation of HBV E / Core Sequence
[0121]A 1.8 Kb BamH I fragment containing the entire precore / core coding region of hepatitis B is obtained from plasmid pAM6 (ATCC No 45020) and ligated into the BamH I site of KS II+ (Stratagene, La Jolla, Calif.). This plasmid is designated KS II+ HBpc / c, FIG. 1. Xho I linkers are added to the Stu I site of precore / core in KS II+ HBpc / c (at nucleotide sequence 1704), followed by cleavage with Hinc II (at nucleotide sequence 2592). The resulting 877 base pair Xho I-Hinc II precore / core fragment is cloned into the Xho I / Hinc II site of SK II+. This plasmid is designated SK+ HBe, FIG. 1.
example 2
Preparation of Sequences Utilizing PCR
A. Site-Directed Mutagenesis of HBV E / Core Sequence Utilizing PCR
[0122]The precore / core gene in plasmid KS II+ HB pc / c is sequenced to determine if the precore / core coding region is correct. This sequence was found to have a single base-pair deletion which causes a frame shift at codon 79 that results in two consecutive in-frame TAG stop codons at codons 84 and 85, FIG. 2. This deletion is corrected by PCR overlap extension (Ho et al., Gene 77:51-59, 1989) of the precore / core coding region in plasmid SK+ HBe. Four oligonucleotide primers are used for the 3 PCR reactions performed to correct the deletion.
[0123]The first reaction utilizes the plasmid KS II+ HB pc / c as the template and as two primers. The sense primer sequence corresponds to the nucleotide sequence 1855 to 1827 of the adw strain and contains two Xho I restriction sites at the 5′ end. The nucleotide sequence numbering is obtained from Genbank (Intelligenics, Inc., Mountain View, Cal...
example 3
A. Isolation of HBV X Antigen
[0219]A 642 by Nco I-Taq I fragment containing the hepatitis B virus X open reading frame is obtained from the pAM6 plasmid (adw) (ATCC 45020), blunted by Klenow fragment, and ligated into the Hinc II site of SK+ (Stratagene, La Jolla, Calif.).
[0220]E. coli (DH5 alpha, Bethesda Research Labs, Gaithersburg, Md.) is transformed with the ligation reaction and propagated. Miniprep DNA is then isolated and purified, essentially as described by Birnboim et al. (Nuc. Acid Res. 7:1513, 1979; Molecular Cloning. A Laboratory Manual. Sambrook et al. (eds.), Cold Spring Harbor Press, 1989).
[0221]Since this fragment can be inserted in either orientation, clones are selected that have the sense orientation with respect to the Xho I and Cla I sites in the SK+ multicloning site. More specifically, miniprep DNAs are digested with the diagnostic restriction enzyme, Bam HI. Inserts in the correct orientation yield two fragments of 3.0 Kb and 0.6 Kb in size. Inserts in the ...
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