An Intracellular Human Single Chain Antibody to Matrix Protein (M1) of H5N1 Virus

Background: Inuenza virus matrix protein M1 is encoded by viral RNA fragment 7 and is the most abundant protein in virus particles. M1 is expressed in the late stages of viral replication and exerts functionality by inhibiting viral transcription. The M1 protein sequence is an attractive target for antibody drugs. Methods: The M1 protein sequence was amplied by RT-PCR using cDNA from the H5N1 virus as a template; the M1 protein was then expressed and puried. A human strain, high anity, and single chain antibody (HuScFv) against M1 protein was obtained by phage antibody library screening using M1 as an antigen. A recombinant TAT-HuScFv protein was expressed by fusion with the TAT protein transduction domain (PTD) gene of HIV to prepare a human intracellular antibody against avian inuenza virus. The differences between HuScFv and TAT-HUScFv were veried by various experiments and the amino acid binding site of the M1 protein was determined. Results: The M1 protein of H5N1, HuScFv, and TAT-HuScFv, were successfully puried and expressed by and in E. coli. Further analysis demonstrated that TAT-HuScFv inhibited the hemagglutination activity of the 300TCID 50 H1N1 virus, thus providing preliminary validation of the universality of the antibody. After two rounds of M1 protein decomposition, the TAT-HuScFv antigen binding site was identied as Alanine (A) at position 239. Collectively, our data describe a recombinant antibody with high binding activity against the conserved sequences of avian inuenza viruses. This intracellular recombinant antibody blocked the M1 protein that infected intracellular viruses, thus inhibiting the replication and reproduction of H5N1 viruses. Conclusion: Recombinant HuScFv was successfully identied using the Tomlinson (I+J) phage antibody library and successfully linked to the TAT protein transductive domain of the HIV virus. Compared with the HuScFv, the addition of the TAT peptide improved its ability to penetrate the cell membrane. A denite amino acid binding site was identied after the decomposition of M1 protein, thus providing a target and reference for the development of antibody drugs and the study of new drugs.


Introduction
The H5N1 virus is a highly pathogenic type A avian in uenza that can cause systemic or respiratory disease in humans (Yee, Carpenter, and Cardona 2009). However, H5N1 is mainly transmitted among birds leads to death. The highly pathogenic H5N1 avian in uenza virus has infected more than 500 million poultry worldwide since human infection with the H5N1 subtype was rst reported in Hong Kong, China, in 1997 (Chen 2009). Subsequently, human infections have emerged in Asia, Europe, and Africa, consequently leading to signi cant public health concerns (Joseph et al. 2017). The 16 sub-types of hemagglutinin HA (H1-H16) and the nine sub-types of neuraminidase NA (N1-N9) are proteins involved in avian in uenza (Noisumdaeng et al. 2021). The combination of different subtypes of HA and NA is responsible for the signi cant diversity of avian in uenza viruses. Page 3/19 The in uenza virus matrix protein is encoded by viral RNA fragment 7; this has two open reading frames and can therefore be transcribed into two mRNAs that are translated into M1 and M2 proteins, respectively (Raman et al. 2020). The M1 protein, with molecular weight of approximately 26 kD, is found extensively in virions, and its sequence is highly conserved. Based on these characteristics, M1 has been used as the basis for classifying in uenza viruses into types A, B and C. M1 protein is expressed in the late stages of viral replication and is concentrated in discrete cell lacunae (Poungpair et al. 2009). A small amount of M1 protein translocates to the nucleus during the late stages of viral infection and helps to inhibit viral transcription. Thereafter, M1 interacts with the NEP (NS2 protein) tail encoded by viral RNA fragment 8 to export vRNP from the nucleus to the cytoplasm ( In this study, a single chain antibody with high a nity was screened by the application of a phage antibody library-Tomlinson I + J based on the M1 protein of the H5N1 virus as a target. In view of the transduction properties of protein transduction domain (PTD)-mediated protein across the cell membrane, the PTD of HIV was used to link to the single-chain antibody molecules (Yin et  The cDNA sequence of the M1 protein for H5N1 virus was identi ed in the NCBI library; this sequence was then used as a template for primer design: forward: 5′-ATG AGT CTT CTA ACC GAG GTC-3′ and reverse: 5′-CCG GAA TTC TTA CTT GAA TCG CTG CAT CTG CAC T-3′. The M1 gene was ampli ed using H5N1 (A/Meerkat/Shanghai/SH-1/2012) cDNA as a template. The PCR reaction conditions were as follows: 94°C denaturation for 5 min, 94°C for 50 s, 50°C for 50 s, 72°C for 50 s (for 34 cycles) followed by 72°C 10 min. The ampli cation products were separated by gel electrophoresis and then ligated into the PET-SUMO vector with T4 ligase. The vector was sequenced and then transformed into BL21 (DE3) competent cells.
Biopanning of a Phage Display Library and Selection by M1 speci city The phage antibody library was prepared in accordance with the manufacturer's instructions and then screened with the puri ed M1 protein as an antigen. During this process, the M1 protein was coated in 96well plates (Nunc-Nalgene, USA) at a concentration of 5µg/ well and then incubated at 4°C overnight. The next day, the supernatant was discarded and washed three times with PBS to remove the non-adsorbed antigen. Non-speci c binding was blocked with 200 µL of 2% milk/PBS at 37°C for 2 hours. After discarding the sealing solution, the wells were washed three times with PBS, and the liquid was removed by vigorous shaking. Next, the Tomlison I+J phage antibody library was added and diluted with 2% milk/PBS to a titer of 1.0 ×10 13 ; 100 µL was added to each well, and the liquid was incubated with vigorous shaking at room temperature for 60 min. After standing for 60 min, the liquid was discarded, and the wells were washed 10 times with PBS (0.05% (V/V) Tween-20). The residual liquid in each well was patted dry and 50µL of eluting solution (5 mg/mL trypsin-PBS) was added to each well. The plates were then shaken at room temperature for 15 min to eluate the phages, which were then stored at 4°C.
The eluded phage was cloned into E. coli TG1 and further panning was performed. Second, third, and fourth rounds of panning were performed under similar conditions, except that the concentration of the antigen coating was reduced to 2 µg/well. Unbound phages were removed by 20, 30, and 40 washes with PBS (0.05% (V/V) Tween-20).
Next, 2% milk/PBS (100µL/Well) was added to the plate, and the plate was kept overnight at 4℃.
Nonspeci c binding was then blocked with 2% BSA/PBS for 2 h and the phage antibody from the fourth round of screening was added. After incubation at room temperature for 1 h, the supernatant was collected to remove the phages that had been speci cally adsorbed to the milk powder in the antibody library; the collected phages were then stored at 4°C.

Expression of Positive Clones and ELISA Analysis for M1 Protein
After four rounds of screening, 10µL of phages were added to 200µL of fresh E. coli HB 2151 and left for 30 min in a water bath at 37°C. Then, 50 µL was applied to a TYE (15 g bacto-agar, 8 g Nacl, 10 g tryptone, 100g Ampicillin, 10g Glucose, 5 g yeast extract in 1L) plate and cultured overnight at 37°C. Once grown on the plate, single colonies were randomly selected and placed on a 96-well culture plate; each well contained 100 µL of 2×TY (30 g bacto-agar, 16 g Nacl, 20 g tryptone, 100g Ampicillin, 10g Glucose, 10 g yeast extract in 1L) medium and cultured overnight at 37°C. The next day, approximately 2 µL of bacterial solution from each well was placed in another 96-well cell plate (the remaining solution was added to glycerol at a nal concentration of 15% and stored at -70°C). The new cell plate contained 200µL of 2×TY medium (containing 100 µg/mL Amp and 0.1% glucose) from each well and cultured at 37°C to an OD 600 of 0.9 (after approximately 4 h of culture). Isopropyl β-D-Thiogalactoside (IPTG) at a nal concentration of 1 mmol/L was added to each well and cultured overnight on a 30°C shaker. After overnight culture, the bacterial solution was centrifuged at 1,800 g for 15 min; the supernatant was then transferred to a new plate and stored at 4°C to await testing. M1 protein (2 µg, 100 µL/well) was added to 96-well plates and incubated overnight at 4°C. The next day, the plates were washed three times (3 min each time) with wash solution (0.05% PBS (V/V) and Tween-20). Next, 200 µL of 2% milk/PBS was added to each well and incubated at 37°C for 1 h. Then, 100 µL of HB2151-induced supernatant was used as a negative control; this was added to each well and incubated at 37°C for 1h. Next, the enzyme label plate was washed three times (for 3min each time) and the excess liquid was patted dry. Next, 100 µL (1:500) of Protein A-HRP was added to each well and incubated at 37°C for 1h. Washing was carried out three more times (3min each time) and excess liquid was patted dry. o-Phenylenediamine (OPD) solution (100 µL) was then added to each well and incubated at room temperature in the light for 20 min. Finally, 2 mol/L of sulfuric acid (50 µL) was added to each well to stop the reaction and the OD 490 absorption value was determined.

Sequence Determination of Selected Phage Clones and the Expression & Puri cation of HuScFv
The M1-positive binding phage in the monoclonal ELISA was used as a template, and vector speci c primers (LMB3: 5 -CAG GAA ACA GCT ATG AC-3 ; PHEN: 5 -CTA TGC GGC CCC ATT CA-3 ) were used to amplify the HuScFv gene fragment. The obtained ampli cation products were subsequently detected by 1% agarose gel electrophoresis. The PCR ampli cation conditions were as follows: pre-denaturation at 94°C for 5 min, 94°C for 50 s, 54°C for 50 s, 72°C for 120 s (35 cycles) and a nal extension at 72°C for 10 min. The target DNA fragment was then recovered and sequenced by Kumei Biological Engineering Co. (China).
ELISA-positive strains were transferred to 5 mL of 2× TY medium containing 100 µg/mL Amp and 1% glucose and cultured overnight at 37°C. The next day, 200 µL of overnight culture was transferred to 2× TY medium (containing 100 µg/mL Amp and 0.1% glucose) and cultured at 37°C to an OD 600 of 0.9 (approximately 4 h). A nal concentration of 1 mmol/L of IPTG was added for overnight induction on a shaking table at a 30°C. On the third day, the induced bacterial solution was centrifuged at 5000 g (Beckman, USA) for 30 min; the supernatant was removed and precipitated with 10%-55% saturated ammonium sulfate. The precipitated solution was then resuspended with 30 mmol/L of PB (pH7.2) and dialysis was performed in PBS overnight. The crude samples were then puri ed by Protein-A FF a nity chromatography; eluted samples were dialyzed with PBS overnight. The target protein was nally analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Western Blotting & Immunoa nity Analysis with HuScFv
The puri ed M1 protein was separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane using a protein electrophoresis transfer device (BIO-RAD) at 45 V for 35min. Membranes were then blotted with HuScFv as a primary antibody and ProteinA-HRP as a secondary antibody (diluted with 1:2,000 PBS). DAB was used as a color reagent to detect the immunobinding activity of the antibody. The binding ability of HuScFv to M1 was determined by a non-competitive ELISA method. Different concentrations of the M1 protein antigen were coated with skimmed milk powder for 1 h, and different dilutions of HuScFv were added. Then, proteinA-HRP and OPD color solution were added; 2 M of sulfuric acid was used as a termination solution, and the absorbance was measured at 490 nm wavelength. A nity constants were calculated using the formula Ka . IgBLAST (a tool for immunoglobulin (IG) and T cell receptor (TR) V domain sequences, NCBI) was used to analyze the nucleotide sequences of clones with the highest immune a nity constant to determine the HuScFv framework and complementary determination region (CDR).

Expression and Puri cation of TAT-HuScFv
Forward (5'-GTG AAT TCA TAA TGA AAT ACC TAT TGC CT-3') and reverse (5 '-GCA AGC TTC TAT GCG GCC CCA TTC AG-3') sequences were introduced into EcoR I and Hind III sites, respectively. A HuScFv plasmid with a high immunoa nity constant was extracted and used as a template for PCR. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min, then 34 cycles of 94°C for 50 s, 50°C for 50 s, and 72℃; this was followed by a nal extension at 72°C for 10 min and cooling at 4°C.
Ampli cation products were recovered by gel electrophoresis. The ampli cation products and the vector (pET28a-TAT-GFP) were extracted by the double digestion of EcoR I and Hind III, respectively; the ampli cation product was then ligated with T4 ligase and transformed into competent E. coli DH5α. Positive clones were then identi ed by PCR.
The pET28A-TAT-HuScFv construct was then transformed into BL21(DE3) with the CaCl 2 method. A single colony was selected and inoculated into LB liquid medium (containing 50 μg/mL Kan) at 37°C and 180 rpm until the OD 600 was approximately 0.5. IPTG was added to a nal concentration of 1 mmol/L and induction was carried out over a culture period of 4 h. The induced bacterial culture was centrifuged at 4°C at 5000g for 20 min, and the bacteria were collected. The bacteria were suspended with TE (pH 8.0) buffer solution and ultrasonically crushed in an ice bath (power: 1500 W; working time: 5 s; interval time: 9 s; 50 times in total). Microscopic examination con rmed that the bacteria had been completely broken down. Centrifugation was carried out at 12,000 g; samples of supernatant and precipitate were then separated by 12% SDS-PAGE.
The supernatant of the expressed cells was obtained following ultrasonic lysis, and a metal chelated Cu 2+ column was used as the buffer system for PBS (pH 7.2). The elution peaks of the target proteins were analyzed with 20 mM and 200 mM imidazole, respectively. The eluent was then puri ed on a rProteinA FF column. Finally, eluted samples were dialyzed with PBS.
Hemagglutination Inhibition Analysis of Anti-M1-HuScFv and TAT-HuScFv Digested MDCK cells were placed in a 96-well cell culture plate (3×10 4 cells/well), and the cells grown into a single layer to be absorbed into the medium. The cells were washed three times with DMEM, and A/Changchun/01/2009 H1N1 was added to each well at different concentrations (PBS was added to the negative control well). The cells were incubated at 37°C for 3.5 h, and the extracellular uid was discarded. Cells were washed twice with PBS; puri ed HuScFv and TAT-HuScFv (10 μg/well; the positive control included PBS only) were then added and incubated at 37°C for 1.5 h. The supernatant of cells was then absorbed and DMEM (containing 1% FBS) was added to each well and cultured overnight at 37°C with 5% CO 2 . The next day, the hemagglutination inhibition test was performed with the overnight culture supernatant. The supernatant was added to the reaction plate (50 μL/well), along with fresh 0.85% chicken red blood cell suspension (50 μL/well), and incubated at room temperature for 30 min; the results of the test were observed by the upright reaction plate.

TAT-HuScFv Bound to Amino Acid Sites of M1 Protein Epitopes
The M1 protein was sequenced and then decomposed in order from the N-terminal to the C-terminal to synthesize 10 polypeptides (Table 1). Positive fragments were detected by the sandwich ELISA method, as described earlier. The polypeptides were used to coat 96-well plates, using TAT-HuScFv as the rst antibody, and protein A-HRP as the second antibody. Analysis involved 3, 3′,5 ,5′-Tetramethylbenzidine (TMB) color and the positive fragments. The positive fragments were decomposed into peptides according to the overlapping sequences of four amino acids and then detected by sandwich ELISA. Positive results were analyzed and positive fragments were synthesized into peptides; these were tested again by sandwich ELISA until the amino acid sites that bound to TAT-HuScFv were identi ed.

Preparation of the H5N1-M1 Protein
The M1 protein (H5N1, A/Meerkat/Shanghai/SH-1/2012) gene was successfully ampli ed and cloned into the pET-Sumo-TAT vector. PCR identi cation revealed a target band at 750 bp (Fig. 1A). Sequencing results showed that the sequence encoding the M1 protein had been cloned into the vector in the correct reading frame. The expression vector was transformed into E. coli BL21 (DE3) and induced with IPTG (at a nal concentration of l mmol/l). The target protein (purity 95%) (Fig. 1B) was successfully obtained and was 30KDa in size.
The Use of a Phage Display Library to Identify HuScFv Speci c to M1 H5N1 Using puri ed M1 protein as an antigen and the Tomlinson (I + J) phage antibody library, we identi ed four strains of anti-M1 protein HuScFv by four rounds of biopanning (1C, 7B, 3F and 4G). The PCR > method was used to determine whether the positive strains with biological activity had been detected successfully and speci c fragments of 930 bp were ampli ed from the four positive strains (Fig. 2A).
Western blotting was performed for the ELISA-positive strains and M1 protein. Analysis showed that 3F and 7B had strong binding ability with the M1 protein and that the stain location and stain depth were more advantageous than other forms of HuScFv under the same conditions (Fig. 2B). A nity constants for 1C, 7B, 3F and 4G were determined by uncompetitive enzyme immunoassay, and a total of four Ka values were obtained according to the simulated biological curve of protein concentration for M1 (Table 2). IgBLAST was used to analyze 3F in the NCBI library to determine the HuScFv framework and CDR region (Fig. 3).

Characterization Of The Recombinant Tat-huscfv Trans-body For M1
The 3F and 7B genes (with the highest immune a nity constant) were successfully cloned into the pET28a-TAT expression vector (Fig. 4) and successfully expressed into BL21 (DE3). SDS-PAGE further showed that the protein product was approximately 28KDa in size (Fig. 5); this was the expected size of the fusion protein. TAT-HuScFv was highly expressed in IPTG-induced E. coli, and was eluted in 200 mmol of imidazole when puri ed by the metal chelated Cu 2+ column. The puri ed TAT-HuScFv and HuScFv were compared for hemagglutination inhibition; the ability of TAT-HuScFv when fused to the TAT domain to bind viral M1 protein was stronger than that of HuScFv (Table 3).

Speci c Recognition Sites Of M1 Protein For Tat-huscfv
ELISA was conducted between puri ed TAT-HuScFv and small peptides based on M1-protein decomposition. Only fragment 10 ( Table 1) was positive; all nine of the other fragments were negative. Because polypeptide number 10 had only 26 amino acid sequences; four of its amino acids were overlapped to synthesize multiple small peptides. After ELISA was conducted again, peptides 5, 6 and 7 were all positive (Fig. 6A). These three small peptides all contained the same two amino acids (LENLQA, NLQAYQ, QAYQKR, QA). Two polypeptides, containing eight amino acids (Q A) were synthesized; then, Q A was genetic mutated into E (ENLEAYQK) G(ENLEQGYQK) respectively. Finally, ELISA (the disordered peptide with the same sequence as the source peptide was also the negative control) was performed (Fig. 6B); peptide 1 was positive and peptide 2 was negative. Therefore, it was inferred that TAT-HuScFv speci cally bound to Alanine (A) at position 239 in the M1 protein.     (TCID50)   50  100  150  200  250  300  350  400   3F  ----+  +  +  +   TAT-3F  ---- does not cause additional in ammation. Typically, each speci c antibody molecule binds to its target using several amino acid residues in the complementary determination region (CDR) and the immunoglobulin framework (FR) of the VH and VL domains. Antibody drugs have an advantage over traditional small-molecule drugs in terms of responding to viral mutations (Ascione et al. 2009

Conclusions
We developed a recombinant HuScFv antibody with high a nity for in uenza virus M1 protein by using