Gclq receptor, HIV-laplzo area jointed with same and relative peptide and target-directional antibody
A carrier, conjugation technology, applied to gClq receptor
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Embodiment 1
[0055] Example 1 Expression of gC1q-R protein in Escherichia coli
[0056] A. Isolation of RNA and preparation of eDNA: According to the manufacturer's recommendation (Biotecx, Houston, Texas) by RNA-ZOL extraction method from 5 × 10 6 Total RNA was isolated from DAKIKI cells. In a solution containing 50 mM Tris-HCl (pH 8.3) and 20 units of RNASIN (Promega, Madison, Wisconsin), 0.5 mM each of dATP, dTTP, dCTP, dGTP, 10 μM oligo dT and 2 units of AMV reverse transcriptase (Gibco BRL, 10 micrograms of RNA was used as template in a reverse transcription reaction mixture at Gaithersburg, Maryland) to prepare the first strand of cDNA. The reaction was carried out at 42°C for 1 hour.
[0057] B. PCR amplification of gC1q-R cDNA encoding mature full-length gC1q-R protein (74 leucine to 282 glutamine of SEQ ID NO: 1):
[0058] The sequences of the two primers used for PCR are from gC1q-R cDNA gene (A.R.Krainer et al., Cell 1991; 66:383-394), primer 1 adds a NdeI restriction site, a...
Embodiment 2
[0067] Example 2 Preparation of monoclonal antibody against gC1q-R peptide
[0068] Twelve-week-old male BALB / cJ mice (Jackson Laboratories, Bar Harbor, Michigan) were injected subcutaneously with 100 μg of pure gC1q-R expressed in E. coli in 200 μl of PBS (Difco Laboratories, Detroit, Michigan). Maine). One month later, mice were injected subcutaneously with 100 μg of gClq-R in Freund's incomplete adjuvant. Mice were injected subcutaneously again one month later with 100 μg of the same antigen in incomplete Freund's adjuvant and sacrificed three days later. For each fusion, single cell suspensions were prepared from spleens of immunized mice and used for fusion with Sp2 / 0 myeloma cells. will be 5×10 8 Sp2 / 0 cells with 5×10 8 Splenocytes were fused in a medium containing 50% polyethylene glycol (molecular weight 1450) (Kodak, Rochester, New York) and 5% dimethylsulfoxide (Sigma Chemical Co., St. Louis, Missouri). Then add 10% fetal bovine serum, 100 units / ml penicillin, 1...
Embodiment 3
[0070] Example 3 Preparation of polyclonal antibody against gC1q-R
[0071] Two male New Zealand White rabbits, approximately 15 weeks old, were immunized subcutaneously with 100 μg of pure gClq-R expressed in E. coli (Difco Laboratories) in complete Freund's adjuvant. They were reinjected two weeks later with the same amount of antigen in Freund's incomplete adjuvant. The same immunization was repeated two weeks later. Sera were collected from immunized animals and tested for reactivity with gClq-R in ELISA as described above, except that bound antibody was detected using HRP-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, ) diluted 1:2000 in BLOTTO . Sera were collected from animals with higher serological responses. Polyclonal rabbit anti-gClq-R immunoglobulin was purified by affinity column using gClq-R coupled Affigel 102 (BioRad Laboratories).
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