Gclq receptor, HIV-1 ph120 region binding thereto, and related peptides and targeting antibodies
An antibody and targeting technology, applied in the direction of carrier binding/immobilization peptide, receptor/cell surface antigen/cell surface determinant, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc.
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Embodiment 1
[0056] Example 1 Expression of gC1q-R protein in E. coli
[0057] A. Isolation of RNA and preparation of cDNA: According to the manufacturer’s recommendations (Biotecx, Houston, Texas) by RNA-ZOL extraction from 5×10 6 Isolate total RNA from two DAKIKI cells. In one containing 50mM Tris-HCl (pH8.3) and 20 units of RNASIN (Promega, Madison, Wisconsin), 0.5mM each of dATP, dTTP, dCTP, dGTP, 10μM oligo dT and 2 units of AMV reverse transcriptase (Gibco BRL, In the reverse transcription reaction mixture of Gaithersburg, Maryland, 10 micrograms of RNA was used as a template to prepare the first strand of cDNA. The reaction was carried out at 42°C for 1 hour.
[0058] B. PCR amplification of gC1q-R cDNA encoding mature full-length gC1q-R protein (leucine 74 to glutamine 282 of SEQ ID NO:1):
[0059] The sequences of the two primers used for PCR are derived from the gC1q-R cDNA gene (ARKrainer et al., Cell 1991; 66: 383-394). Primer 1 adds an NdeI restriction site, and primer 2 adds a Ps...
Embodiment 2
[0068] Example 2 Preparation of monoclonal antibodies against gC1q-R peptide
[0069] 100 μg of E. coli expressing pure gC1q-R in 200 μl PBS (Difco Laboratories, Detroit, Michigan) was subcutaneously injected into 12-week-old male BALB / cJ mice (Jackson Laboratories, Bar Harbor, Maine). One month later, mice were injected subcutaneously with 100 μg of gClq-R in Freund's incomplete adjuvant. One month later, the mice were subcutaneously injected with 100 μg of the same antigen in Freund's incomplete adjuvant, and sacrificed three days later. For each fusion, a single cell suspension was prepared from the spleen of immunized mice and used for fusion with Sp2 / 0 myeloma cells. 5×10 8 Sp2 / 0 cells and 5×10 8 Each splenocyte was fused in a medium containing 50% polyethylene glycol (molecular weight 1450) (Kodak, Rochester, New York) and 5% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, Missouri). Then Iscove medium (Gibco, Grand Island, New York) supplemented with 10% fetal bovine ser...
Embodiment 3
[0071] Example 3 Preparation of anti-gC1q-R polyclonal antibody
[0072] Two male New Zealand white rabbits about 15 weeks old were subcutaneously immunized with 100 μg of pure gClq-R (Difco Laboratories) expressed in E. coli in Freund's complete adjuvant. Two weeks later, they were injected again with the same amount of antigen in Freund's incomplete adjuvant. Repeat the same immunization two weeks later. Serum was collected from immunized animals and tested for reactivity with gC1q-R in ELISA as described above, except that HRP-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories,) diluted 1:2000 in BLOTTO was used to detect bound antibodies . Serum is collected using animals with higher serological responses. The polyclonal rabbit anti-gClq-R immunoglobulin was purified by an affinity column using gClq-R coupled Affigel 102 (BioRad Laboratories).
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