發(fā)布時(shí)間：2018-05-02 23:27

  本文選題：豬圓環(huán)病毒2型 + 微載體懸浮培養(yǎng)��； 參考：《中國(guó)農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：豬圓環(huán)病毒2型及其相關(guān)性疫病已成為危害養(yǎng)豬業(yè)的主要疫病之一,疫苗是防控PCV2的有效工具之一,但目前商品化疫苗存在諸如有效抗原含量低、免疫應(yīng)激大等弱點(diǎn),因此開發(fā)具有抗原含量高、純度高、應(yīng)激小、免疫劑量少等優(yōu)勢(shì)的PCV2疫苗,是今后的研究熱點(diǎn)之一。本研究旨在利用細(xì)胞懸浮培養(yǎng)技術(shù)、抗原純化技術(shù)和滅活凍干技術(shù),研究PCV2-ZJ/C株的規(guī)模化培養(yǎng)工藝、抗原高效純化工藝,并在此基礎(chǔ)上,研制了PCV2凍干滅活疫苗,為PCV2凍干滅活疫苗的研究與臨床應(yīng)用提供參考。PCV2 ZJ/C株微載體懸浮培養(yǎng)工藝研究：采用有限稀釋法對(duì)PK15細(xì)胞在微載體上進(jìn)行馴化培養(yǎng)PK15細(xì)胞,獲得了微載體培養(yǎng)適應(yīng)性PK15細(xì)胞克隆株(命名為PK-15-S株),在此基礎(chǔ)上,利用微載體生物反應(yīng)器研究PK1 5細(xì)胞懸浮培養(yǎng)參數(shù)和PCV2-ZJ/C株的懸浮培養(yǎng)參數(shù)。結(jié)果表明,PCV2-ZJ/C株病毒滴度為108.3-108.5TCID50/mL,相當(dāng)于傳統(tǒng)轉(zhuǎn)瓶培養(yǎng)工藝的6.3-10倍。PCV2 ZJ/C株懸浮培養(yǎng)病毒液的純化工藝研究：采用澄清、濃縮、層析純化等技術(shù)組合,對(duì)病毒滴度為108.3-108.5TCID50/mL的懸浮培養(yǎng)病毒液進(jìn)行純化,獲得高純度的病毒蛋白,并按照中華人民共和國(guó)農(nóng)業(yè)部第1893號(hào)公告中的豬圓環(huán)病毒2型滅活疫苗(ZJ/C株)的質(zhì)量標(biāo)準(zhǔn)進(jìn)行免疫效力檢驗(yàn),結(jié)果表明,當(dāng)純化抗原的總蛋白濃度為20有效PCV2蛋白濃度0.34μg/mL時(shí),免疫保護(hù)率在80%以上。PCV2凍干滅活疫苗的制備：采用凍干技術(shù)和抗原耐熱保護(hù)技術(shù),設(shè)計(jì)10種耐熱保護(hù)劑(B1-10)和3種凍干曲線,優(yōu)化凍干曲線和耐熱保護(hù)劑,批量生產(chǎn)了3批疫苗。按照現(xiàn)有凍干疫苗的質(zhì)量標(biāo)準(zhǔn)和農(nóng)業(yè)部第1893號(hào)公告中的豬圓環(huán)病毒2型滅活疫苗(ZJ/C株)的質(zhì)量標(biāo)準(zhǔn)進(jìn)行耐熱穩(wěn)定性、免疫效力和保存期研究,結(jié)果表明,3批PCV2凍干滅活苗的各項(xiàng)指標(biāo)均符合規(guī)程要求；37℃條件下保存14天,2-8℃保存期至少18個(gè)月,具備良好的臨床應(yīng)用前景。
[Abstract]:Porcine circovirus type 2 and its associated blight have become one of the major diseases that harm the pig industry. Vaccine is one of the effective tools to prevent and control PCV2. However, the commercialized vaccine has such weaknesses as low effective antigen content and high immune stress. Therefore, the development of PCV2 vaccine with high antigen content, high purity, low stress and low immune dose is one of the research hotspots in the future. The purpose of this study was to study the scale culture and high efficiency purification of PCV2-ZJ/C strain by cell suspension culture technique, antigen purification technique and inactivated freeze-drying technique. On this basis, a freeze-dried PCV2 vaccine was developed. To provide a reference for the research and clinical application of PCV2 freeze-dried inactivated vaccine. The suspension culture technology of PK15 cell line ZJ/C strain was studied. PK15 cells were domesticated and cultured on microcarrier by finite dilution method. An adaptive PK15 cell clone (named PK-15-S strain) was obtained, and the suspension culture parameters of PK1 5 cell and PCV2-ZJ/C strain were studied by microcarrier bioreactor. The results showed that the virus titer of PCV2-ZJ / C strain was 108.3-108.5 TCID _ (50) / mL, which was equivalent to 6.3-10 times of the traditional culture technology. The purification process of suspension culture of PCV2-ZJ / C strain ZJ/C strain: clarification, concentration, chromatographic purification, and so on. The suspension culture solution with 108.3-108.5TCID50/mL titer was purified to obtain high purity virus protein. In accordance with the quality standard of inactivated porcine circovirus type 2 vaccine ZJ / C strain, published by Ministry of Agriculture No. 1893 of the people's Republic of China, the results showed that when the total protein concentration of purified antigen was 0.34 渭 g/mL, the total protein concentration of purified antigen was 0.34 渭 g/mL. Preparation of freeze-dried vaccine with immune protection rate of more than 80%: 10 kinds of heat-resistant protective agents (B1-10) and 3 kinds of freeze-dried curves were designed by using freeze-drying technique and antigenic heat-resistant protection technique. The freeze-drying curve and heat-resistant protectant were optimized and three batches of vaccines were produced in batches. According to the quality standards of the existing freeze-dried vaccine and the quality standard of the inactivated porcine circovirus type 2 vaccine strain ZJ / C in announcement No. 1893 of the Ministry of Agriculture, the heat stability, immune efficacy and preservation life of the vaccine were studied. The results showed that all the indexes of three batches of PCV2 freeze-dried seedlings were in accordance with the requirements of regulations. The storage period of 14 days and 8 鈩，

本文編號(hào)：1835934