Cloning And Expression Of Porcine Interferon Gamma Gene And Studies On The Anti-PRRSV Effect Of Joint Application Of Recombinant Protein And Chinese Herbal Medicines

Interferons (IFNs) have many kinds of biologic activities such as anti-virus, regulate immune function. Its immediate effect and multi-activities make it playing an important role on preventing and curing viral infectious diseases, promoting animal growth and so on in animal husbandry. Mature porcine interferon gamma (PoIFN-γ) was cloned and expressed in E.coli expression system in this study. The Chinese herbal medicines which can inhibit the PRRSV proliferating were screened by CPE and MTT methods, and then the inhibitory effect of joint application of PoIFN-γand Chinese herbal medicines to PRRSV was evaluated.1. Cloning and expression of porcine interferon gamma geneA pair of primers were designed according to the porcine interferon gamma gene in GenBank. Then the PoIFN-γgene was amplified from porcine spleen's total RNA by reverse transcription chain reaction (RT-PCR), agarose gel electrophoresis result indicated its size about 500 bp, match the theory size. The PCR product was purified and inserted into E.coli clone vector pMD18-T and then one of the positive recombinant clones was sequenced and analyzed. Sequencing result showed that the homology was 99% between the gene we cloned and the one published in GenBank, point mutation happened, nucleotide from G mutated to A, and the corresponding amino acid from arginine changed to histidine, but all were alkality polar amino acid. The analyzed result from the internet showed that the theory molecular weight of recombinant protein was 18.033 KD, the theory isoelectric point was 9.54.The PoIFN-γgene was subcloned into a prokaryotic expression vector pET-28a(+), thus, recombinant expression plasmid PoIFN-γ/pET-28a(+) was constructed. The recombinant plasmid PoIFN-γ/pET-28a(+) was transformed into E.coli BL21 cells, and induced by IPTG (final concentration was 1 mmol/L) at 37℃for 4 hours. The recombinant porcine IFN-γwas expressed efficiently in the form of inclusion body, SDS-PAGE result showed that the molecular weight of recombinant protein was about 20 KD and the yield accounting for 37.2% of total bacterial proteins.Western-Blot analysis of the gel showed a band of similar size was the major protein immunoreactive with the anti-6×His MAb. After the recombinant PoIFN-γwas purified, renaturation and concentrated, its antiviral activity was assayed by inhibition of the cytopathic effect (CPE) of Vesicular Stomatitis Virus (VSV) on Marc-145 cell line. Result showed the antiviral titre of recombinant PoIFN-γwas about 1.3×10³U/mL (5.0×10³ U/mg). This study we finished the groundwork for studying on the anti-PRRSV effect of joint application of recombinant PoIFN-γand Chinese herbal medicines. 2. Studies on the anti-PRRSV effect of joint application of recombinant protein and Chinese herbal medicinesIn this study, Marc-145 cells were cultured in vitro. The TC₀ and TC₅₀ of twelve Chinese herbal medicines, Radix Isatidis, Flos Chrysanthemi Indici, Herba Andrographitis, Radix Astragali, Flos Lonicerae, Herba Houttuyniae, Radix Bupleuri, Rhizoma Coptidis, Fructus Forsythiae, Radix Glycytthizae, Radix Scutellariae, Herba Violae, were determined by CPE and MTT methods. And the Chinese herbal medicines which can inhibit the PRRSV proliferating were screened, simultaneously, the IC₅₀ and TI of Chinese herbal medicines were also calculated.The TC₀ and IC₅₀ of the twelve Chinese herbal medicines on Marc-145 cells were as follow: Radix Isatidis, 31.25 mg/mL, 1.38 mg/mL; Flos Chrysanthemi Indici, 7.81 mg/mL, 1.30 mg/mL; Herba Andrographitis, 7.81 mg/mL, 0.91 mg/mL; Radix Astragali, 3.90 mg/mL, 0.22 mg/mL; Flos Lonicerae, 31.25 mg/mL, 2.08 mg/mL; Herba Houttuyniae, 3.90 mg/mL, 0.44 mg/mL; Radix Bupleuri, 15.63 mg/mL, 2.54 mg/mL; Rhizoma Coptidis, 3.90 mg/mL, 0.35 mg/mL; Fructus Forsythiae, 7.81 mg/mL, 1.28 mg/mL; Radix Glycytthizae, 15.63 mg/mL, 2.62 mg/mL; Radix Scutellariae, 0.98 mg/mL, 0.13 mg/mL; Herba Violae, 31.25 mg/mL, 2.47 mg/mL. And the IC₅₀ of recombinant PoIFN-γwas 99.50 U/mL. The three Chinese herbal medicines which the TI were ahead of other medicines, were Radix Astragali, Radix Scutellariae and Radix Isatidis, the TI were 70.22, 56.47 and 44.66 individually.By the evaluation of Chinese herbal medicines' anti-PRRSV effects and developing value. Three Chinese herbal medicines, Radix Astragali, Radix Scutellariae and Radix Isatidis, were selected, and the inhibitory effect of joint application of recombinant PoIFN-γand Chinese herbal medicines to PRRSV was evaluated.The combine index (CI) and Isobologram analysis results showed that the anti-PRRSV activities of recombinant PoIFN-γand Chinese herbal medicines (Radix Astragali, Radix Scutellariae and Radix Isatidis) were significantly higher than that of recombinant PoIFN-γor Chinese herbal medicines alone.