CN102321653B - Applications of man-mouse chimeric CD4 (cluster of differentiation 4) plasmid and multiclonal antibody in inhibiting HIV (human immunodeficiency virus) infection - Google Patents

Abstract

The invention discloses a human-mouse chimeric CD4 plasmid, the sequence of which is shown in SEQ ID No.1. The plasmid preparation method of the present invention is simple and convenient. Experiments have confirmed that the plasmid constructed by the present invention can effectively mediate HIV virus infection by co-transfecting 293T cells with co-receptors. The inhibition rate of HIV pseudoviruses is 60-90%, showing good broad-spectrum neutralization ability.

Description

Application of Human-mouse Chimeric CD4 Plasmid and Its Polyclonal Antibody in Inhibiting HIV Infection

technical field

The invention relates to a chimeric plasmid, specifically a human-mouse chimeric CD4 plasmid, and a polyclonal antibody prepared from the plasmid.

Background of the invention

In the past 20 years, research results in the field of HIV-1 vaccines have shown that both T cell-mediated cellular immunity and B cell-mediated humoral immunity are indispensable through research on HIV-1, SIV and SHIV. Antibodies can protect the body from infection by viruses (such as measles virus, hepatitis virus, and influenza virus, etc.), but for highly mutated viruses like HIV-1, it is difficult to obtain antibodies with broad-spectrum neutralizing activity through immunization . HIV-1 neutralizing antibody targets are mainly viral membrane protein trimers and susceptible target cell surface receptors to prevent HIV-1 virus from infecting target cells. Although HIV-1 evades the host immune system through membrane protein mutations, masking of glycosylation sites, interactions between proteins, and conformational masking of important functional regions, there are still six broad-spectrum neutralizing antibodies that pass recognition The HIV-1 membrane protein is produced from a relatively conserved region. Compared with the highly variable HIV-1 membrane protein, the highly conserved characteristics of the cell receptor CD4 and its co-receptor will become an important target for therapeutic antibodies in clinical trials and the key for HIV-1 to enter cells. It has been reported that the CCR5 inhibitor maraviroc only prevents R5-tropic HIV-1 from infecting target cells, but is ineffective against X4 and amphotropic viruses. Antibody research targeting CXCR4 is more difficult. During the recent HIV vaccine research, studies on CD4 monoclonal antibody have shown that it has a strong ability to neutralize multi-subtype virus strains. CD4mAb ibalizumab (i.e. TNX-355 and Hu5A8) is a humanized IgG4 monoclonal antibody that has shown antiviral activity in phase I and phase II clinical trials. In this paper, by constructing human CD4 and human-mouse chimeric plasmid DNA to immunize mice, the monoclonal antibody with broad-spectrum neutralization ability was obtained through the screening of multiple antiserum and monoclonal antibody antiviral ability. Hope to provide more theoretical and experimental basis for future preventive and therapeutic vaccines.

Contents of the invention

The purpose of the present invention is to provide a human-mouse chimeric CD4 plasmid.

The second object of the present invention is to provide the polyclonal antibody prepared by utilizing the above-mentioned plasmid and having obvious inhibitory effect on HIV pseudovirus.

Another object of the present invention is to provide the use of the polyclonal antibody.

The inventive idea of the present invention is: HIV infects target cells first by binding to its surface CD4 receptor and co-receptor, so if the combination of HIV and CD4 receptor can be blocked, HIV infection can be effectively inhibited. Human-mouse chimeric plasmids in the V1V2 region of CD4 receptors are expected to obtain polyclonal antibodies with HIV-inhibiting functions through DNA immunization, providing theoretical and experimental basis for exploring new targets for HIV vaccine and drug development.

The present invention adopts following concrete technical scheme:

A human-mouse chimeric CD4 plasmid, the sequence of which is shown in SEQ ID No.8.

Application of the above-mentioned plasmid in the preparation of medicines for mediating HIV virus infection.

The above-mentioned preparation method of human-mouse chimeric CD4 plasmid, said method comprises the steps:

(1) Total RNA was extracted from Ghost cells and mouse splenocytes respectively;

(2) Use P1 and P2 to amplify human total RNA to obtain human CD4, and P3 and P4 to amplify mouse total RNA to obtain mouse CD4;

(3) P3, P10 amplify mouse CD4 to obtain the mouse CD4 signal peptide; P11, P7 amplify human CD4 to obtain the human CD4V1V2 region sequence; use P3, P7 to amplify the mouse CD4 signal peptide and human CD4V1V2 region to obtain the mouse CD4 signal peptide and human Chimeric product of CD4V1V2 region;

(4) The product obtained in step (3) is amplified through three rounds of PCR to finally obtain the human-mouse chimeric CD4 plasmid; the first round of PCR amplification primers are P3, P8, and the second round of PCR amplification primers are P9, P4 , The primers for the third round of PCR amplification are P3 and P4.

The PCR conditions in the step (3) are: 94°C for 5min; 94°C for 30s, 55°C for 40s, 72°C for 1min, 20 cycles; 72°C for 8 minutes.

The PCR conditions in the step (4) are: 94°C for 5 minutes; 94°C for 40s, 55°C for 50s, 72°C for 90s, 25 cycles; 72°C for 8 minutes.

A method for preparing a human-mouse chimeric CD4 polyclonal antibody, said method comprising the steps of: using the above-mentioned human-mouse chimeric CD4 plasmid DNA to immunize 4-week-old BALB/C mice, respectively at the 1st and 21st 100μL 1mg/mL of the plasmid DNA described in claim 1 was injected intramuscularly into both hind limbs of mice on days 35 and 49, and the injection site was electrically stimulated with a 5mm wide double-electrode needle at 60V. Each electrical stimulation lasted for 50-ms, a total of 6 times. Mouse serum was collected on day 10.

The human-mouse chimeric CD4 polyclonal antibody prepared by the above method.

Use of the above-mentioned polyclonal antibody in the preparation of drugs for inhibiting HIV pseudoviruses.

A eukaryotic expression vector is constructed by using the above-mentioned plasmid and vector pcDNA3.1/V5-His-TOPO.

Beneficial effects of the present invention:

The plasmid preparation method of the present invention is simple and convenient. Experiments have confirmed that the plasmid constructed by the present invention can effectively mediate HIV virus infection by co-transfecting 293T cells with co-receptors. The inhibition rate of HIV pseudoviruses is 60-90%, showing good broad-spectrum neutralization ability.

The practicability, outstanding substantive features and positive effects of the present invention can be manifested through the following examples, but the scope thereof is not limited.

Description of drawings

Figure 1 is the amino acid sequence alignment of hCD4, mhD1D2m, mhD2m and mCD4. The eukaryotic expression plasmids pcDNA3.1-hCD4, pcDNA3.1-mhD1D2m, pcDNA3.1-mhD2m and pcDNA3.1-mCD4 were constructed and sequenced, and the amino acid sequences of human CD4, human-mouse chimeric CD4 and mouse CD4 were compared.

Fig. 2 shows the inhibitory effect (A) and the specific binding ability (B) to human CD4 of the polyclonal antibody prepared by immunization of human-mouse chimeric plasmid DNA against HIV-1 pseudovirus. CD4 binding ability FACS detection Hela negative control cells and negative control serum reaction curve before immunization are shown in black, serum reaction curves of Hela negative control cells and human mouse chimeric plasmid after immunization are shown in red, TZM-bl positive cells and negative control before immunization are shown in red The control serum reaction curve is shown in green, and the serum reaction curve after TZM-bl positive cells immunized with human-mouse chimeric plasmid is shown in blue.

Detailed ways

The construction of embodiment 1 human-mouse chimeric CD4 plasmid

1. Use TRIzol from Ghost cells (Ghost cells are known cells, can be purchased, and are HOS cells stably expressing human CD4, CCR5 and CXCR4) and mouse splenocytes (cells are derived from fresh mouse splenocytes) respectively. Extract total RNA; the method is as follows:

Extraction of total cellular RNA by TRIzol method

(1) For about 10 ⁶ cells, use RNase-free tips to add 1mL TRIzol to make it work fully and pipette;

(2) Let it stand at room temperature for 5 minutes, then transfer it to an RNase-free 1.5mL centrifuge tube;

(3) Add 0.2 mL of chloroform, pipette for 15 s, and let stand at room temperature for 3 min;

(4) Centrifuge at 12,000 rpm for 15 minutes at 4°C, and the solution can be seen to be divided into three phases. The upper colorless aqueous phase contains RNA; the middle phase contains DNA; the lower red phenol/chloroform phase contains protein;

(5) Absorb the aqueous phase into a new 1.5mL centrifuge tube;

(6) Add 0.5mL of isopropanol, let it stand at 4°C for 10min, and at the same time add 10mL of 75% ethanol, that is, 7.5mL of absolute ethanol plus 2.5mL of RNase-free water;

(7) Centrifuge at less than 12,000 rpm for 10 minutes at 4°C, and discard the supernatant;

(8) Add 1mL of 75% ethanol, and evenly precipitate;

(9) Centrifuge at less than 7500rpm at 4°C for 5min, and discard the supernatant;

(10) Put the 1.5mL centrifuge tube containing the precipitate into a 37°C incubator, and dry the precipitate;

(11) Add 13 μL of RNase-free water, bathe in water at 57°C for 10 minutes, and dissolve the precipitate;

(12) Add 1 μL of RNase-free DNase, and bathe in water at 37°C for 20 minutes to remove impurity DNA;

(13) Put it in a 4°C refrigerator for later use.

Use P1 and P2 to amplify human total RNA to obtain human CD4, and P3 and P4 to amplify mouse total RNA to obtain mouse CD4; the first step of RT-PCR reaction conditions: 42°C, 30min; the second step reaction conditions: 94°C for 5min; 94 Denaturation at ℃ for 50s, annealing at 55℃ for 1min, extension at 72℃ for 2min, 35 cycles; extension at 72℃ for 10min. The finally obtained human CD4 nucleotide sequence is shown in SEQ ID No.1; the finally obtained mouse CD4 nucleotide sequence is shown in SEQ ID No.2.

2. Using primers P3, P10, P11, P7, P8, P9, and P4 (see Table 1 for primer sequences), amplify the chimeric product of mouse CD4 signal peptide and human CD4 V1V2 region by overlapping PCR;

2.1 Using mouse CD4 as a template, use primers P3 and P10 to PCR amplify the signal peptide of mouse CD4 to obtain the sequence shown in SEQID No.3. PCR reaction conditions: 94°C for 5 minutes; 94°C for 30s, 55°C for 40s, 72°C for 1 minute, 20 cycles; 72°C for 8 minutes.

2.2 Using human CD4 as a template, use P11 and P7 PCR to amplify the V1V2 region of human CD4 to obtain the sequence shown in SEQ ID No.4); PCR reaction system 50 μl, see Table 1; PCR reaction conditions: 94°C for 5 minutes; 94°C for 30 seconds , 55°C annealing for 40s, 72°C extension for 1 minute, 20 cycles; 72°C extension for 8 minutes.

2.3 Use P3 and P7 to amplify the products obtained in the above two steps to obtain human-mouse chimeric CD4, the sequence of which is shown in SEQ ID No.5; PCR reaction system 50 μl, see Table 1; PCR reaction conditions: 94 ° C for 5 minutes ; 94°C for 30 seconds, 55°C for 40 seconds, 72°C for 1 minute, 20 cycles; 72°C for 8 minutes.

3. Obtain the full length of human-mouse chimeric CD4 by overlapping PCR (the V1V2 region of human CD4 replaces the V1V2 region of mouse CD4)

3.1P3, P8 Amplify the mouse CD4 signal peptide obtained in step 2.3 and the chimeric product of human CD4 V1V2 region, and the obtained sequence is shown in SEQ ID No.6; 50 μl of PCR reaction system, see Table 1. PCR reaction conditions: 94°C for 5 minutes; 25 cycles of 94°C for 40s, 55°C for 50s, 72°C for 90s, and 72°C for 8 minutes.

3.2 Using SEQ ID No.6 as a template, use P9 and P4 PCR to amplify the mouse CD4 V3V4 region sequence, and finally obtain the sequence shown in SEQ ID No.7; see Table 1 for 50 μl of PCR reaction system. PCR reaction conditions: 94°C for 5 minutes; 25 cycles of 94°C for 40s, 55°C for 50s, 72°C for 90s, and 72°C for 8 minutes.

3.3 Using the product obtained in the above two steps as a template, use primers P3 and P4 to PCR amplify to obtain the full-length sequence of human-mouse chimeric CD4, and finally obtain a human-mouse chimeric CD4 plasmid. The sequence of the plasmid is as shown in SEQ ID No. 8.

50 μl of PCR reaction system, see Table 1. PCR reaction conditions: 94°C for 5 minutes; 25 cycles of 94°C for 40s, 55°C for 50s, 72°C for 90s, and 72°C for 8 minutes.

Table 1 PCR reaction system (50μL)

3.4 Combine the obtained human-mouse chimeric CD4 with the vector pcDNA3.1/V5-His-TOPO to construct the plasmid pcDNA3.1-mhD1D2m

The obtained human-mouse chimeric CD4 and the carrier pcDNA3.1/V5-His-TOPO were digested by HindIII and XhoI respectively, then electrophoresed on 1% agarose gel, recovered by cutting the gel, and connected to obtain the true Nuclear expression plasmid pDNA3.1-mhD1D2m.

The primer sequences used are shown in Table 2

Table 2

Example 2 Preparation of human-mouse chimeric CD4 polyclonal antibody

The constructed plasmid can effectively mediate HIV virus infection by co-transfecting 293T cells with co-receptor CCR5.

pcDNA3.1-mhD1D2m plasmid DNA was extracted using Qiagen EndoFree Plasmid Maxi Kits (Qiagen, Chatsworth, CA).

After immunizing 4-week-old BALB/C mice (5 mice/group) with plasmid DNA, intramuscularly inject 100 μL of 1 mg/mL plasmid DNA of the present invention into the hind limbs of the mice on the 1st, 21st, 35th, and 49th days respectively, with a 5mm wide double-electrode needle The injection site was electrically stimulated with 60V, and each electrical stimulation lasted 50-ms, a total of 6 times. The mouse serum was taken as a negative control before immunization, and the mouse serum was collected 10 days after each immunization for the detection of antiviral function (HIV-1 sham Virus neutralization assay) or binding capacity assay (FACS).

Example 3 Human-mouse chimeric CD4 polyclonal antibody inhibits different subtypes of HIV-1 pseudoviruses

The pseudovirus neutralization experiment refers to the reduction of the expression level of the luciferase reporter gene under the action of the inhibitor after the pseudovirus infects the Ghost cells, and is used to evaluate the neutralization ability of the inhibitor.

Preparation of pseudovirus: pHEP-ENV membrane protein plasmid and pNL4-3Luc RE-backbone plasmid (1g:4g) were transiently transfected into 293T cells. After transfection, the 293T cells were incubated for 48 hours at 37°C in a 5% CO ₂ incubator. The pseudoviruses in the supernatant were harvested and stored at -80°C.

Pseudovirus infection and detection: inoculate 1× ¹⁰⁴ Ghost cells in each well of a 96-well plate, 80% of the cells form a layer the next day, inactivate the serum sample at 56°C for 1 hour before the experiment, and mix the polyclonal antibody serum sample with the cells after doubling dilution and pseudoviruses were co-incubated at 37°C, and the expression of luciferase (RLU) was detected after 48 hours. Calculate percent neutralization or ID50.

Flow cytometry analysis: 1×10 ⁶ TZM-b1 cells were incubated with 1:100 diluted serum for 1 h on ice, Hela cells were used as negative control, after washing with PBS, FITC-labeled goat anti-mouse secondary antibody diluted 1:50 was incubated on ice After incubation for 1 h, the cells were washed twice with PBS, and analyzed by flow cytometry (Becton-Dickinson, USA)

The inhibitory rate of polyantiserum obtained from pcDNA3.1-mhD1D2m plasmid DNA immunization against HIV-1 pseudovirus infection in Ghost cells was systematically evaluated by different subtypes of HIV-1 pseudovirus (Chinese strain). The multiple antisera used to neutralize each pseudovirus subtype were diluted, and the results showed that the antisera obtained after immunization with pcDNA3.1-mhD1D2m plasmid DNA were more effective against various strains of different subtypes. With strong inhibition rate, the ID ₅₀ of polyantiserum after immunization with pcDNA3.1-mhD1D2m was 540 (Fig. 2A).

After pcDNA3.1-mhD1D2m immunization, the specific binding of polyantiserum to CD4 on the cell surface was further confirmed by FACS detection of TZM-bl and Hela cells. As shown in Figure 2B, the pre-immune serum had no binding to Hela and TZM-bl cells. After immunization, the polyantiserum (diluted 100 times) has a significant difference in binding to Hela and TZM-bl cells respectively, that is, when the polyantiserum obtained is diluted 100 times, it still has a strong specific binding ability to human CD4 on the cell surface .

Claims (9)

1. a people mouse mosaic type CD4 plasmid, is characterized in that, the sequence of described plasmid is as shown in SEQ ID No.8.

2. the application of the described plasmid of claim 1 in preparation mediation HIV virus infective medicament.

3. the preparation method of people mouse mosaic type CD4 plasmid claimed in claim 1, is characterized in that, described method comprises the steps:

(1) from Ghost cell and mouse boosting cell, extract total RNA respectively;

(2) utilize P1, P2 amplification human total rna to obtain people CD4, the total RNA of P3, P4 amplification mouse obtains mouse CD4;

(3) P3, P10 amplification mouse CD4 obtains mouse CD4 signal peptide; P11, P7 amplification people CD4 obtains people CD4V1V2 region sequence; Utilize P3, P7 amplification mouse CD4 signal peptide and people CD4V1V2 district to obtain mouse CD4 signal peptide and the chimeric product in people CD4V1V2 district;

(4) product that step (3) is obtained finally obtains people mouse mosaic type CD4 plasmid through the three-wheel pcr amplification; First round pcr amplification primer is P3, P8, and second to take turns the pcr amplification primer be P9, P4, and third round pcr amplification primer is P3, P4;

Described primer P1 sequence is as shown in SEQ ID No.9, primer P2 sequence is as shown in SEQ ID No.10, primer P3 sequence is as shown in SEQ ID No.11, primer P4 sequence is as shown in SEQ ID No.12, primer P7 sequence is as shown in SEQ ID No.13, and primer P8 sequence is as shown in SEQ ID No.14, and primer P9 sequence is as shown in SEQ ID No.15, primer P10 sequence is as shown in SEQ ID No.16, and primer P11 sequence is as shown in SEQ ID No.17.

4. the preparation method of people mouse mosaic type CD4 plasmid claimed in claim 3, is characterized in that, the PCR condition is in described step (3): 94 ℃ 5 minutes; 94 ℃ of 30s, 55 ℃ of annealing 40s, 72 ℃ were extended 1 minute, 20 circulations; 72 ℃ were extended 8 minutes.

5. the preparation method of people mouse mosaic type CD4 plasmid claimed in claim 3, is characterized in that, the PCR condition is in described step (4): 94 ℃ 5 minutes; 94 ℃ of 40s, 55 ℃ of annealing 50s, 72 ℃ are extended 90s, 25 circulations; 72 ℃ were extended 8 minutes.

6. a carrier for expression of eukaryon, is characterized in that, it utilizes plasmid claimed in claim 1 and carrier pcDNA3.1/V5-His-TOPO to build and forms.

7. the preparation method of a people mouse mosaic type CD4 polyclonal antibody, it is characterized in that, described method comprises the steps: that right to use requires 6 described carrier for expression of eukaryon immunity BALB/C mice in 4 age in week, respectively the 1st, 21,35, the 49 days two hindlimb muscle injection 100 μ L1mg/mL of mouse plasmid DNA claimed in claim 1, the wide two electrodes pin of 5mm 60V electricity irritation injection site, each electricity irritation continues 50-ms, and totally 6 times, immunity zoomed in mouse serum in latter 10 days.

8. the people mouse mosaic type CD4 polyclonal antibody for preparing of method claimed in claim 7.

9. polyclonal antibody claimed in claim 8 suppresses the purposes in HIV pseudovirus medicine in preparation.

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