鸡是我国各地区广泛养殖的主要家禽，但鸡在养殖过程中容易受到各种病毒如传染性法氏囊病毒、新城疫病毒、和禽流感病毒的感染而死亡，给养殖户带来巨大的经济损失。疫苗是防治鸡传染病的最有效手段，因此针对各种鸡致病病毒研发新型疫苗对保护我国养鸡产业具有非常重要的意义。但目前我国疫苗生产以传统的生产工艺进行生产，其技术落后于国外先进技术。鸡传染性法氏囊炎是传染性法氏囊病毒（Infections bursal disease virus，IBDV）入侵鸡的淋巴器官法氏囊而引发的急性传染病，传染性和死亡率都非常高。

本研究旨在运用CRISPR/Cas9系统敲除鸡胚成纤维细胞的TLR3受体基因，降低细胞因TLR3参与的细胞对病毒的免疫拮抗作用，从而提升病毒在宿主细胞中的增殖含量。进而研究IBDV在基因敲除后的宿主细胞中的增殖情况，并建立该细胞克隆株的微载体悬浮培养及病毒规模化增殖工艺，为IBDV抗原的规模增殖提供生物材料并奠定工程技术积累。取得以下成果：

（一）TLR3敲除鸡胚成纤维细胞DF-1-TLR3-KO的构建

采用CRISPR/Cas9技术成功地构建敲除了TLR3序列的鸡胚成纤维细胞。筛选出的DF-1-TLR3-KO#4单克隆细胞的IBDV病毒增殖效价比在DF-1原始细胞中增加了16.7%，为IBDV的大规模增殖提供了技术基础。

（二）DF-1-TLR3-KO细胞微载体悬浮培养及IBDV增殖工艺的建立

确定了DF-1-TLR3-KO细胞的最佳接种密度为3.0×105个/mL，与之适配的微载体cytodex1 最佳添加量为 3g/L，感染复数（Multiplicity of Infection，MOI）为0.05时IBDV增殖效果最好。相比于常规批式培养，换液和补料均可以显著提高DF-1-TLR3-KO细胞密度，其中换液操作效果更佳，细胞培养密度比常规批次培养提高约91%，比补料培养提高约16%。在5L反应器中进行的5个批次换液操作试验中增殖的IBDV效价均超过8.0 lg TCID50/mL。

（三）5L生物反应器中DF-1-TLR3-KO细胞微载体悬浮培养不同工艺比较

在5L生物反应器中研究了消化传代放大、分批换液和循环灌注式等3种不同的工艺路线对DF-1-TLR3-KO细胞增殖影响。（1）分批换液操作工艺条件下，3g/L的微载体使用量即可获得适宜的细胞培养效果，获得最大的DF-1-TLR3-KO活细胞密度达到23.8×105个/mL。（2）消化传代放大操作工艺条件下，6g/L的微载体使用量获得最大的DF-1-TLR3-KO活细胞密度达到28×105个/mL，利用胰酶消化并不会导致细胞死亡。（3）循环灌注式操作工艺条件下，6g/L的微载体使用量获得最大的DF-1-TLR3-KO活细胞密度达到60×105个/mL，显著高于分批换液和消化传代操作工艺。同时，循环灌注培养方式获得的IBDV病毒增殖效价最高达到了9.5 lgTCID 50/mL，比分批换液和消化传代操作工艺的病毒增殖滴度高出约10倍。

（四）DF-1-TLR3-KO细胞微载体培养增殖IBDV的模拟放大

利用田口正交实验，确定了DF-1-TLR3-KO细胞培养的消化传代工艺：消化液浓度0.5g/L、EDTA 0.4g/L、胶原酶0.2g/L，在35rpm搅拌转速下作用10min。初步设计出DF-1-TLR3-KO细胞微载体悬浮培养、消化传代放大、IBDV接毒增殖的全过程。整个5L～60L规模 DF-1-TLR3-KO细胞放大培养及IBDV增殖过程控制在12天左右，生产周期较短且操作简便、易于规模化应用。

Chickens are the main poultry widely cultivated in China. However, chickens are susceptible to viruses such as infectious bursal virus, Newcastle disease virus and avian influenza virus, which bring huge economic losses to farmers. Vaccines are the most effective means to prevent and control chicken infectious diseases. Therefore, the research and development of new vaccines against various chicken-causing viruses is of great significance. However, at present, China's vaccine production is produced by traditional production technology with low efficiency and low quality. Infectious bursal disease of chickens is an acute infectious disease caused by infectious bursal disease virus (Infections bursal disease virus, IBDV). The infectivity and mortality of IBD are very high. The purpose of this study is to knock out the TLR3 receptor gene of chicken embryo fibroblasts using CRISPR/cas9 system to reduce the immune antagonism and improve the proliferation of IBDV in host cells. Then the proliferation of IBDV in the knockout cells was studied, and the microcarrier suspension culture and virus large-scale proliferation process were established. The main results are as follows:

1. Construction of TLR3 gene knockout chicken embryo fibroblast cell DF-1-TLR3-KO

Chicken embryo fibroblasts with TLR3 gene knockout were successfully constructed by CRISPR/cas9 technology. The proliferation titer of IBDV in DF-1-TLR3-KO#4 cells was 16.7% higher than that in non-knockout cells.

2. Establishment of suspension culture of DF-1-TLR3-KO and IBDV proliferation 

The optimum inoculation density of DF-1-TLR3-KO cells was determined to be 3.0×105 cells/mL, the optimal addition of microcarrier cytodex1 was 3g/ L, and the optimal MOI was 0.05. Compared with conventional batch culture, fluid change and feeding could significantly improve the density of DF-1-TLR3-KO cells. The effect of fluid change was better that feeding. The cell culture density acquired in fluid change process was about 91% higher than that of conventional batch culture and 16% higher than that of feeding culture. The titer of proliferated IBDV exceeded 8.0 lg TCID50/mL in 5 batches of liquid change operation tests in 5L reactor.

3. Comparison of different processes of DF-1-TLR3-KO cell suspension culture in 5L bioreactor

The effects of three different processes on the proliferation of DF-1-TLR3-KO cells were studied in a 5L bioreactor. (1) Under the batch liquid exchange process, the optimal amount of microcarrier was 3g/L, and the maximum cell density of DF-1-TLR3-KO reached 23.8 × 105 cells/mL. (2) Under the digestion process, the optimal amount of microcarrier was 6g/L, and the maximum cell density of DF-1-TLR3-KO reached 28× 105 cells/mL. (3) Under the circulating perfusion process, the optimal amount of microcarrier was 6g/L, and the maximum cell density of DF-1-TLR3-KO reached 60× 105 cells/mL, which was significantly higher than the batch liquid exchange and digestion processes. Furthermore, the proliferation titer of IBDV reached 9.5 lgTCID 50/mL under circulating perfusion process, which was about 10 times higher than that of batch exchange and digestion processes.

4. Simulated amplification of IBDV proliferation in DF-1-TLR3-KO cell microcarrier culture

The digestion process of DF-1-TLR3-KO cell culture was determined using Taguchi orthogonal experiment: digestion solution concentration = 0.5g/L, EDTA 0.4g/L, and collagenase 0.2g/L, incubation for 10min at 35rpm. The whole process of DF-1-TLR3-KO cell microcarrier suspension culture, digestion, and IBDV inoculation and proliferation in 5L～60L reactor scale were preliminarily designed.