Preparation and bioactivity of anti-Newcastle disease virus-phosphoprotein cytoplasmic transduction peptide antibody

On the basis of cell-penetrating peptide’s character that it can penetrate cytomembrane and transfer macromolecular protein to cytoplasm so to play biological function, we took the experiments. The fuse penetrating peptide our experiment adoptted is HIV-TAT derived fragment-CTP512, with good transmember effect and distinct cytoplasm-position. In this chapter, the research of transmembrane character was processed first. According to the tests on trans- member protein with different concentrations, the best trans-member concentration is 3µM. Afterwards, we found that the location of trans-member antibody is overlapping with phosphoprotein using indirect immunofluorescence test analysis. According to MTT test, there is no significant difference between CTP fusion protein and control on cell proliferation and viability. TCID50 test was used to detect the protective effect of transmember antibody on cell. Result showed that trans- member antibody has significant cell protection effect compared to the control in the order: ZL.103>ZL.17>Control. Fluorogenic quantitative PCR result showed that trans- member antibody can disturb the duplication and transcription of Newcastle disease virus. This results not only paved a good way to research the transport of disease related protein, but also provide a splendid tool on protein function research. using specific primers for P (sense 5’- and antisense 5’-GTTTTGCCTTGTGGGATTGC-3’) andβ-actin (sense 5’-GCATCCACGAAACTACATTCAACTC-3’ and antisense 5’-CACTGTGTTGGCATAGAGGTCTTTG-3’), and SYBR dsDNA binding Real-time PCR reaction was performed at 95 ℃ for 3 min for 1 cycle and then 94 ℃ for 45 s, 55 ℃ for 30 s and 72 ℃ for 1 min for 40 cycles. PCR products were measured with Rotor-Gene 2000 Real-time Cycler and analyzed with Rotor-gene software. Cycles times (Ct) were analyzed at a reader of 0.2 fluorescence unit. The duplicate cycle times were averaged and normalized to the cycle time of β-6

light on the research of antibody [5,6]. The mechanism of the entrance to cell of cell-penetrating peptide is unclear, however, its appearance supply a good method to biotherapy and scientific research. Cell-penetrating peptide is a kind of micro-molecule peptide which able to carry external material into cell [7]. So far, it is reported TAT, VP22, MPG, Pep-1 etc. as cell-penetrating peptide [8].
Among them HIV-TAT and its derived fragment are the most popular used cell-penetrating peptide [9,10]. However, because of the nuclear localization of TAT, which function fusing to protein is influenced [11,12]. By transforming the order and structure of HIV-TAT's amino acid, Kim built a new type cytoplasm transmit peptide(CTP), which own good cell member-penetrating character and marked cytoplasm-position feature [13]. As we know, Newcastle disease virus phosphoprotein is located in cytoplasm, so it is advisable to research the interaction of trans-member antibody and phosphoprotein.
In this paper, we built chicken original cytoplasm-expressed gene infused with CTP sequence, which might conduct trans-member transport of antibody in cell. After penetrating of cytomembrane, the antibody will realize the cytoplasm-position in accordance with the location sequence. (So to build valid delivery system of trans-member antibody).

Construction of cytoplasmic transduction peptide-single chain antibody fusion protein
Anti-phosphoprotein scFvs have been cloned from the spleen of immunized chicken in our laboratory, referred to in our publication Biologicals [14]. The CTP was added to 3' terminal. The forward primer was designed as 5'-CGGAATTCGCCGTGACGTTGGAC-3' with EcoR I; and the reverse primer was 5'-GAGTCATTCTGCGGCCGCACGGCGACGCTGGCGACGTTTCTTACGACCGTATAGGACGGTCAGGGTTGTCCC-3' with Not I and CTP. The anti-P CTP-scFv gene was amplified by PCR. after digested with EcoR I and Not I, the CTP-scFv gene was subcloned into the pET28a(+). In this way, the recombinant expression plasmid pET28a-CTP-scFv was obtained.

Stable expression of pET28a-scFv-CTP
Trans1-Blue transfected with the recombinant plasmid pET28a-scFv-CTP was propagated at 37℃ in Luria Bertani (LB) medium supplemented with 100 mg/ml Kanamycin until the bacterium reached logarithmic growth phase (at OD600=0.7), and then induced by addition of 1mM isopropyl b-dthiogalactopyranoside. Purification of the His-tagged phosphoprotein was carried out in accordance with the manufacturer's procedures for Ni+-NTA resin-packed columns. The final protein concentration was determined by the BCA kit.
The best contration of transduce of pET28a-scFv-CTP fusion protein BHK21 cell were passaged (and immigrated) into 6 pore plate. That is : Cells in 100ml cell culture bottle were washed twice using DMEM, after 1min's digestion using tyrisin, cells were blown and suspended in 4mLDMEM(10% FBS with penicillin-streptomycin), and were immigrated into 6 pore plate (0.6ml cell suspension per pore), then were cultured in incubator overnight. When cell's density reached to 80%, cells were treated with protein in different concentrations respectively as following:

MTT test to detect CTP influence on cell viability
Cells were collected in logarithmic phase, and were made into 10 5 /ml suspension using DMEM 5 containing 10% FBS, then were inoculated in 96 culture plate. After 24h, pET28a-scFv-CTP and scFv were treated to cell with final concentration 0.5mg/ml, DMEM was contrast. Every sample owned 4 holes as parallel. Cells were cultured for in 37℃ and 5%CO2. After 24h, cells were treated with 20µl 5mg/ml MTT soluted in PBS , after incubating for 4h, supernatant were removed, and the culture plate was inverted on several layers filter papers so to wipe put supernatant sufficiently. Added DMSO 150 µl per hole, the cells were shaked in shaker for 30min. The OD value were detected in 570nm/490nm using microplate reader.

Virus infection
To assess the effect of anti-P CTP-scFv antibody on NDV production, BHK21 cells were stably transduced with pET28a-scFv-CTP fusion protein, respectively. Then infected with the virus at a multiplicity infection of 0.01, supernatants of cell cultures were harvested at 12, 24, 36 and 48h.

Construction of pET28a-scFv-CTP
The product of RT-PCR coincided with what we preconceived. A desired PCR product of 783 bp was obtained after agarose gel electrophoresis analysis. Then the phophoprotein gene was digested by the double restriction enzyme and further cloned into pET28a(+) vector. The recombinant plasmid pET28a-P was successfully constructed, and the enzyme digestion analysis was identical with the expected results (Fig1. A).

Expression of anti-P pET28a-scFv-CTP
In order to obtain the purified pET28a-scFv-CTP fusion protein, the constructed prokaryotic expression plasmid of pET28a-scFv-CTP was transformed into Trans1-Blue and inducted by 1mM IPTG. A high level of expression protein was obtained. The expression fusion protein with molecular weight of about 33 KD was detected by SDS-PAGE (Fig1. B). The recombinant protein was purified through Nichelating affinity chromatography, and the purity was above 85% by SDS-PAGE gel scan analysis (Fig1. C).

Trans-membrance activity of pET28a-scFv-CTP fusion protein
Immunofluorescence studies were carried out to identify transduced pET28a-scFv-CTP fusion proteins could penetrate in cells. The results showed the the purified pET28a-scFv-CTP fusion proteins have efficient trans-membrance activity and the best contration of transduce of pET28a-scFv-CTP fusion protein is 3µM

Subcellular localization of transducted ZL.17 and ZL.103
Cellular localization of fusion protein ZL.17 and ZL.103 were investigated. BHK21 cells were transducted with the purified fusion protein ZL.17 and ZL.103 carrying an His tag. Results showed the anti-P fusion protein ZL.17 and ZL.103 were distributed uniformly throughout the cell(in both cytoplasm and nucleus) (Fig 1.D).

pET28a -scFv-CTP fusion protein influence on cell viability
MTT assay was performed to reveal whether the fusion protein were harming to BHK21 cells. The 7 result indicated that they were found no cytotoxic effect of pET28a-scFv-CTP fusion protein to BHK21 cells (Fig 2).

Anti-P pET28a-scFv-CTP fusion protein affected NDV F48E9 virus production
Stable transducted cells lines were then infected with the virus at a multiplicity infection of 0.01. 24 h, 36 h, and 48 h post-infection, supernatants of cell cultures were harvested, serially diluted, and assayed for virus titer using the TCID 50 method. Results revealed that virus titers were reduced in cell lines stably expressing all the three anti-P CTP intrabodies, in the order of: ZL.17> ZL.103>control when compared to that of empty vector transfected cells. These results suggested that virus production was potently inhibited by ZL.17 and ZL.103 (Fig 3).

Anti-P pET28a-scFv-CTP inhibitied viral transcription and replication
In order to determine the effect of anti-p intrabodies on viral transcription and replication, we measured the production of three different forms of NDV RNA (mRNA, cRNA and vRNA), by using an approach described previously [16]. Total RNA was isolated at 4 h post-infection when sufficient viral RNA transcription and replication had occurred. Viral mRNA, cRNA, and vRNA were reverse transcribed using corresponding primers and the quantity of cDNA products measured by real-time PCR. Data from the real-time PCR showed that the levels of P-specific mRNA were significantly decreased in the presence of ZL.17 and ZL.103. The levels of P-specific cRNA were significantly inhibited by two anti-P CTP intrabodies. In addition, the P-specific vRNA production was also inhibited by two anti-P intrabodies in the order: ZL.17 ZL.103 Control (Fig 4). These results suggested that anti-P CTP intrabodies have inhibitory effects on the production of NDV mRNA, cRNA and vRNA.

Discussion
CTP is a kind of antibody protein with fusion expression of transport region [17,18]. By means of/Via transmembrane function, CTP can conduct the transmembrane transport of antibody(expressed protein), and then located in cytoplasm [19,20]. As this antibody is protein itself, it need not to be expressed in host cell, so the security is guaranteed in most extent. The consist of cell penetrating peptides is multiple, that it composes of 7-16 amino acid oligopeptides [8]. Generally, there is no or little homology in primary structure and secondary structure of penetrating peptides, however, the 8 common feature is that penetrating peptide is rich in amino acid residue, especially arginine, which has the interaction with the of negative pole in cell surface [21]. Cell penetrating peptide has low toxicity, and besides biomacromolecules such as polypeptide, protein and nucleic acid, it can carry moleculer drug, eikonogen and so on, hence, penetrating peptide is well used in cell research.
So far, it has not been reported that, by means of protein prokaryotic expression and purification technology, combined with cytoplasm transduce peptide fuse strategy, extrinsic protein peptide was transduced to the cytoplasm of BHK21 cell, and to bind with phosphoprotein specifically, so to inhibit the replication and transcription of phosphoprotein in Newcastle disease virus. It's a good tool in researching the transport of disease related protein, also a good method in studying protein function.
In past 20 years, the research of cell-penetrating peptide supplies a promising way in surmounting biological membrane barrier. In 1988, HIV-1Tat protein was firstly reported to be able to get through cytomembrane, soon afterwards, the antimmnpedia transcription protein in drosophila was reported to possess the similar transcription characters [22,23]. The transduction ability of small peptide sequence to these transcription factor can be realized after the other macromolecule such as protein penetrating cell [24]. Many cell-penetrating peptides have been identified so far [25,26]. These peptides become Trojan horse or protein transducing region at first, then turn to cell-penetrating peptide through internalization after reappraising, with showing endocytosis and assisting conduction, is a kind of primary internalization pathway [27]. Among them, the prior found protein-HIV-TAT and its derived fragment are well studied penetrating peptide. However, penetrating peptide has no specificity on tissues and cells, hence, the aiding method is needed in drug delivery system.
According to the character that the phosphoprotein in Newcastle disease virus participates in transcription and replication of RNA, and located in cytoplasm after infecting cell, the fused penetrating peptide our experiment adopted was HIV-TAT derived fragment-CTP512, with good transmembrane effect and distinct cytoplasm-position [10,28,29].
At present, the unsolved problem in administration route is how to transmit therapeutic biomacromolecule penetrating cytomembrane. The confronting problem is how to conquer the low bioavailability caused by biomacromolecule's size and water-wet behavior and the subsequent 9 restriction on transmembrane. Now the problem become increasingly sharp, instead of biomacromolecule, more new drugs have to penetrate one or more lipid bilayer to reach the target spot. Many researches have testified the high efficiency of cell-penetrating peptide in carrying aim polypeptide in vitro. Schwarze has proved the transmembrane effect of cell-penetrating peptide in vitro that via the fusion of penetrating peptide to β-galactosidase, the protein transmit has realized by intraperitoneal injection in mouse [30]. Hence, cell-penetrating peptide is a promising tool in macromolecule delivery.
In this paper, we took the bioactivity analysis of transmembrane fusion protein we acquired. At first, whether pET28a-scFv-CTP protein can entrance BHK21 cell was detected. Result showed that pET28a-scFv-CTP fusion protein was succeed entrancing cell and being expression with concentration dependency that 3 µM is the optimum concentration. Cellular localization under fluorescence microscope found that transmembrane-antibody mainly distributed in cytoplasm, and was co-located with phosphoprotein of Newcastle disease virus. By means of TCID50 test analysis, the two transmembrane-antibodies were capable to protect cell from the infection of Newcastle disease virus.
Fluorogenic quantitative PCR analysis found that, transmembrane-antibody can disturb the production of cRNA, vRNA and mRNA which related to transcription and replication of virus, so to inhibit virus's transcription and replication.

Conclusion
Our experiment verified that, the transmembrane fusion antibodies we built not only develop efficient transmembrane character, but also conduct interest protein to target spot so to exert bioactivity.
According to current report that penetrating peptide does not possess tissues and cells targeting property, and pharmacokinetics and body distribution studies have not been carried out, so there is certain distance from its clinical application [7,31]. However, the results imply a new way in prevention and treatment of Newcastle disease, also provide basis to screening drug targeting