Human heavy chain antibody expression in filamentous fungi
A heavy chain and filamentous technology, applied in the field of expressing human heavy chain antibodies in filamentous fungi, can solve the problem of low solubility of human heavy chain proteins
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Embodiment 1
[0152] Construction of Aspergillus strain Jal355:
[0153] BECh2 is described in WO 00 / 39322, WO 00 / 39322 which further refers to patent WO 98 / 12300 (describing JaL228).
[0154] pJaL173 is described in WO 98 / 12300
[0155] pJaL335 is described in WO 98 / 12300
[0156] In order to remove the defective pyrG gene located in the alkaline protease gene in Aspergillus oryzae bacterial strain Bech2, carry out following operation:
[0157] Isolate pyrG - Aspergillus oryzae strain - ToC1418:
[0158] Aspergillus oryzae strain Bech2 was screened for resistance to 5-fluoro-orotic acid (FOA) to identify spontaneous pyrG mutants. One strain (ToC1418) was identified as pyrG - . ToC1418 is uridine dependent and thus can be transformed with the wild type pyrG gene and transformants can be selected for their ability to grow in the absence of uridine.
[0159] pyrG + Construction of the Aspergillus oryzae strain - JaL352:
[0160] A mutation in the defective pyrG gene located within th...
Embodiment 2
[0175] Construction of plasmids for expression
[0176] In order to promote the expression of the target gene located on the expression plasmid, it is necessary to reduce the expression of the marker gene used for selection (here, the pyrG gene is taken as an example). The total expression level of the selected gene is brought to the level necessary for survival by culturing host cells with an expression plasmid containing a selected gene whose expression has been reduced under normal selective pressure, resulting in selection for host cells with increased plasmid copy number . However, higher plasmid copy numbers also lead to enhanced expression of the gene of interest.
[0177] One way to reduce the expression level of a selected gene is to reduce mRNA levels by using a transcriptionally weaker promoter or reducing the functional half-life of the mRNA. Another way is to reduce the translation efficiency of mRNA. One way of doing this is to mutate the Kozak region (Kozak M...
Embodiment 3
[0261] Herceptin is a human antibody used to treat breast cancer. It is a very expensive product, and similar products would be cheaper to produce in filamentous fungi with high expression potential.
[0262]A gene was constructed based on the amino acid sequence of the human heavy chain fragment of Herceptin with the same codon habits as the highly expressed gene found in Aspergillus.
[0263] In order to be able to synthesize the gene encoding the variable region of the heavy chain of Herceptin, the primers shown in Fig. 1 were synthesized based on the above DNA sequence. The relative positions of the primers are shown in Figure 1.
[0264] Construction of pENI2716:
[0265] Primers 230402j3 (10 pmol), 230402j4 (2 pmol), 230402j7 (10 pmol) and 230402j8 (2 pmol) were mixed in a total volume of 20 μl, and a PCR reaction was carried out using TGO polymerase and buffer (Roche) (94° C. for 5 minutes, ( 94°C for 30 seconds, 50°C for 30 seconds, 72°C for 1 minute) 25 cycles, 72°...
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